EMBO/SourceData huggingface dataset:
Figure 11Spot tests of co-transformants of either Bd0367 (DgcA; GGDEF protein) response regulator domain or Bd3125 (CdgA; a degenerate GGDEF protein likely involved in c-di-GMP binding and signalling) with full length Bd1971, or the N-terminal (1-145), or C-terminal (145-400) portions of Bd1971 in either pUT18c or pKT25 as indicated. Positive control was co-transformants with pUT18c-zip and pKT25-zip, negative control was the empty vectors. The co-transformants were plated on LB-X-Gal medium with positive interaction resulting in blue colouration, which can be seen for all of the interactions in both plasmid combinations, but not for the negative control. Images are representative of at least 4 independent experiments.Confirmation of BTH interactions by β-Galactosidase activity assays. The interaction is significantly stronger between Bd3125 and the N-terminal domain of Bd1971 than the interaction with the C-terminus of Bd1971. The response regulator domain of Bd0367 showed significant interaction with Bd1971, but the full length Bd0367 did not. *p>0.05 by Student's t-test N/S- not significant. Data are from at least three independent experiments, error bars are the standard error of the mean
Figure 1B. Blue native-PAGE loading of protein extracts from nrc2/3/4 knockout N. benthamiana plants after immunoprecipitation with anti-Flag antibody. Co-migration of Rpi-amr3-HF and AVRamr3-V5 are indicated (*, red). Same samples were loaded twice on one blue native-PAGE gel, transferred onto one membrane, and then the membrane was cut into two and immunoblotted separately. GUS-V5, β-glucuronidase fused with V5 tag. C. NRC2EEE-Myc does not alter association between Rpi-amr3 and AVRamr3. Samples of Fig 1B were SDS-boiled and loaded on SDS-PAGE. Input samples were taken prior to immunoprecipitation to show expression of all proteins.
Figure 2A. NRC2EEE-Myc is oligomerized upon effector-dependent activation of Rpi-amr3. Protein lysates from Fig 1B, C were loaded on blue native-PAGE. SDS-boiled protein lysate samples serve as control for actual size of NRC2EEE-Myc. Oligomerized NRC2EEE-Myc is indicated (*, red). B. NRC2EEE-Myc oligomerizes upon effector-dependent activation of Rpi-amr1. Protein lysates from nrc2/3/4 knockout N. benthamiana plants were loaded on blue native-PAGE. Oligomerized NRC2EEE-Myc is indicated (*, red).C. Samples from Fig 2B were SDS-boiled and loaded on SDS-PAGE. Protein accumulation of Rpi-amr1-Flag, NRC2EEE-Myc, AVRamr1-V5 and AVRamr3-V5 are shown.
Figure 3B. NRC2EEE and Rpi-amr3 migration in the absence of effector. N. benthamiana nrc2/3/4 knockout plants were transiently infiltrated with Rpi-amr3-HF, NRC2EEE-Myc and GUS-V5 followed by 2D-PAGE. NRC2EEE-Myc proteins (*, red) and Rpi-amr3-HF proteins (*, blue) is indicated. C. NRC2EEE and Rpi-amr3 migration in the presence of effector. N. benthamiana nrc2/3/4 knockout plants were transiently infiltrated with Rpi-amr3-HF, NRC2EEE-Myc and AVRamr3 -V5 followed by 2D-PAGE. NRC2EEE-Myc proteins (*, red) and Rpi-amr3-HF protein (*, blue) is indicated.D. Re-localization of NRC2EEE-Myc upon effector-dependent activation of Rpi-amr1. Lysates of nrc2/3/4 knockout plants transiently expressing Rpi-amr1-Flag, NRC2EEE-Myc with AVRamr3-V5 or AVRamr1-V5 were fractionated into total (T), soluble (S), and pellet (P) fractions. E. Re-localization of NRC2EEE-Myc upon effector-dependent activation of Rpi-amr3. Lysates of nrc2/3/4 knockout plants transiently expressing Rpi-amr3-HF, NRC2EEE-Myc with AVRamr3-V5 or AVRamr1-V5 were fractionated into total (T), soluble (S), and pellet (P) fractions.
Figure 4A. P-loop of Rpi-amr3 is required for AVRamr3-dependent HR in N. benthamiana. Representative leaf phenotype of HR (hypersensitive response) in wild-type N. benthamiana is shown. The number of leaves tested and occurrences of HR are indicated in parentheses. B. P-loop of NRC2 is required for AVRamr3-dependent HR in N. benthamiana nrc2/3/4 knockout plants. Representative leaf phenotype of HR is shown. The number of leaves tested and occurrences of HR are indicated in parentheses.C. NRC2EEE-Myc requires functional P-loop of both Rpi-amr3 and NRC2 for oligomerization. Protein lysates from N. benthamiana nrc2/3/4 knockout plants expressing wild-type or P-loop mutants of Rpi-amr3 or NRC2EEE were loaded for blue native-PAGE. Membranes were immunoblotted with anti-Myc.
Figure 5B. Protein lysates expressing NRC4AAA-Myc with Rpi-amr3-HF or Rpi-amr1-Flag in the presence and absence of cognate effector were loaded on blue native-PAGE. Proteins were transiently expressed in N. benthamiana nrc2/3/4 knockout plants. Non-specific bands are indicated with *.
Figure 6B. NRC2EEE-Myc oligomerizes in response-dependent manner. Protein lysates from N. benthamiana nrc2/3/4 knockout transiently expressing NRC2EEE-Myc, Rpi-amr3-HF and AVRamr3 alleles from P. infestans, P. capsici, and P. parasitica were loaded on blue native-PAGE and blotted for NRC2EEE (anti-Myc). C. Recognition is correlated with interaction and protein complex formation of AVRamr3 with Rpi-amr3. Protein extracts from Fig 5B were immunoprecipitated with anti-Flag antibody, separated on blue native-PAGE, and AVRamr3-GFP proteins of P. infestans, P. capsici, and P. parasitica were visualized.
figf1(a) HeLa cell lysates were immunoprecipitated with Atg16L1 antibody (Atg16L1) or no antibody (No Ab) in control, and subjected to SDS-PAGE; proteins were stained with SimplyBlue safe stain (Invitrogen) in accordance with the manufacturer's protocol. Bands indicated in the box were cut out and digested with trypsin, and identified by MALDI-TOF MS. HC, heavy chain.(b) HeLa cells transfected for 24 h with control vector, or wild-type or different deletion mutants (residues 2-77, 2-275 and 232-607) of Flag-Atg16L1 were immunoprecipitated with anti-Flag antibody (Atg16L1) and immunoblotted with anti-clathrin, anti-Flag and anti-Atg5 antibodies. Clathrin interacts with the N terminus of Atg16L1, in a similar manner to Atg12-Atg5. Total lysates were run alongside as controls for protein input. FL, full-length.(c) HeLa cell lysates were immunoprecipitated (IP) with Atg16L1 antibody (+Ab) or no antibody (no Ab) and immunoblotted with anti-clathrin, anti-AP2 and anti-Atg16L1 antibodies. Total cell lysates (TL) were also blotted for input controls. Endogenous Atg16L1 interacts with clathrin heavy chain and AP2.(d) HeLa cells transfected for 24 h with Flag-Atg16L1 and increasing concentrations of Atg5 were immunoprecipitated (IP) with anti-Flag antibody (Atg16L1) and immunoblotted with anti-clathrin, anti-Flag and anti-Atg5 antibodies. Atg5 overexpression neither interferes with nor increases clathrin-Atg16L1 binding. Uncropped images of blots are shown in Supplementary Information, Fig. S9.
figf2(a) HeLa cells transfected for 5 days with two rounds of control, epsin 1 (eps.), clathrin heavy chain (cla.), AP2 or AP1 siRNA were either left untreated or treated for 4 h with bafilomycin A1 (+/- BafA1), after which they were lysed for western blot analysis with anti-LC3 and anti-tubulin antibodies. Note that LC3-I is often very faint compared with LC3-II in HeLa cells under the protein extraction conditions we use. However, this is not a problem because it is advisable to relate LC3-II to tubulin/actin.(b) Ratio of LC3-II to tubulin on knockdown (KD) of control, epsin, clathrin heavy chain, AP2 or AP1, with or without bafilomycin A1, quantified from three independent experiments (two for epsin) and represented in the graph. Asterisk, P 0.01; two asterisks, P 0.001; three asterisks, P 0.0001.(c) HeLa cells stably expressing GFP-mRFP-LC3 (ref. 17) were transfected for 5 days with two rounds of control or clathrin heavy chain siRNA, during which they were either left untreated (UT) or treated with bafilomycin A1 (BafA1) for the last 15 h. Cells were then fixed and analysed on a Cellomics ArrayScan system. Quantification of autophagic vacuoles (AV) or autolysosomes (AL) per cell in the different conditions is shown in the graph. Asterisk, P 0.01; n.s., not significant. n = 2,000 cells.(d) HeLa cells transfected for 4 days with control, clathrin heavy chain, AP1 or AP2 siRNA were grown in Hanks balanced salt solution (HBSS) for 6 h, after which the cells were fixed and immunostained for endogenous LC3. The percentages of HeLa cells with more than 50 LC3 vesicles (marked with arrows) are quantified in the graph. End., endogenous. Three asterisks, P 0.0001. n = 500 cells. All error bars represent s.e.m. Scale bars, 10 μm. Uncropped images of blots are shown in Supplementary Information, Fig. S9.
figf3(a-d) HeLa cells transfected for 72 h with control, epsin 1, clathrin heavy chain, AP2 or AP1 siRNA were treated with HBSS (to induce autophagy) for 6 h, after which they were fixed and immunostained for endogenous Atg16L1. Representative cells for the control and epsin 1 siRNA are in a, and percentages of HeLa cells with Atg16L1vesicles are quantified in b (for epsin 1), c (for clathrin) and d (for AP1 and AP2). Three asterisks, P 0.0001. Scale bars, 10 μm. (Note that we used single siRNAs for all experiments and the effects were confirmed with two independent sequences for clathrin heavy chain). n = 600 cells.(e, f) HeLa cells transfected for 24 h with Flag-tagged wild-type Atg16L1 or the Atg16L1 deletion mutants 2-275 Atg16L1 or 232-607 Atg16L1 were fixed and immunostained with anti-Flag antibody (e); the proportions of cells with Flag-Atg16L1 vesicles are represented in f. Representative images of Atg16L1 vesicles with the different mutant constructs are shown in e. The large boxes in each panel are higher magnifications of regions in the smaller boxes. Three asterisks, P 0.0001; n.s., not significant. n = 100 cells.(g-i) HeLa cells transfected with control, epsin 1, clathrin heavy chain, AP2 or AP1 siRNA, as above, were transfected with GFP-Atg16L1 and tomato LC3 for a further 24 h, after which the cells were fixed. g shows representative cells for control, epsin 1 and clathrin siRNA. The percentage of GFP-Atg16L1 (green) that co-localized (yellow, marked by arrows) with tomato LC3 vesicles (red) is quantified as shown in the graph in h (control, epsin 1 and clathrin siRNA) and i (control, AP2 and AP1 siRNA). Three asterisks, P 0.0001. n = 21 cells. Scale bars, 10 μm. All error bars represent s.e.m.
figf4(a) HeLa cells transfected for 24 h with GFP-Atg16L1 were fixed to reveal the Atg16L1 vesicles (green). Scale bar, 10 μm.(b) HeLa cells transiently transfected for 20 h with GFP-Atg16L1 and mRFP-GPi were cultured in starvation medium for a further 2 h. TIRF image with GFP-Atg16L1 (green) and mRFP-GPi (red) is shown. Arrows indicate co-localization between Atg16L1 and GPi. The percentage co-localization of GFP-Atg16L1 with mRFP-GPi is shown in the graph. n = 2 cells. Scale bar, 5 μm.(c) HeLa cells transfected for 24 h with GFP-Atg16L1 were either left untreated or treated with 50 μM dynasore (Sigma) for 4 h and processed for immunogold electron microscopy with anti-GFP antibodies (15-nm gold particles) and anti-clathrin antibodies (10-nm gold particles). Co-localization can be seen in the boxed areas. Quantification of clathrin-coated structures (CCS) that were associated with Atg16L1 per 1,000 μm2 is shown in the graph. Three asterisks, P 0.0001. Scale bar, 100 nm. All error bars represent s.e.m.
figf5(a) HeLa cells transfected for 24 h with GFP-Atg16L1 (green) and CFP-LC3 (blue) were incubated with Alexa Fluor-555-conjugated cholera toxin subunit B (red) for 15 min at 4 °C (allowing toxin to bind to the plasma membrane). Then cells were incubated at 37 °C (allowing internalization of cholera toxin) for 10 min and fixed for confocal analysis. Vesicles positive for both Atg16L1 and cholera toxin are yellow (also see high-magnification images at the right). Note that the small Atg16L1 vesicles co-localizing with cholera toxin are negative for LC3 (as marked with yellow arrows), and the Atg16L1 vesicles co-localizing with both cholera toxin and LC3 (marked with blue arrows) are shown in the magnified panels at the right. Scale bar, 10 μm.(b) HeLa cells transfected for 24 h with GFP-Atg16L1 (green) were incubated with Alexa Fluor-555-conjugated cholera toxin subunit B (red) as in a, after which they were fixed, immunostained for endogenous (end.) EEA1 (blue) and analysed by confocal microscopy. Vesicles positive for Atg16L1 and cholera toxin B are yellow (white arrows) and vesicles positive for both EEA1 and cholera toxin are purple (blue arrows) and are shown in the high-magnification images in the middle. The graph shows percentage co-localization. Cho. tox., cholera toxin. n = 20 cells. Scale bar, 10 μm.(c) HeLa cells treated with HBSS for 6 h were incubated with Alexa Fluor-555-labelled cholera toxin subunit B (red) as in a. Cells were then fixed, and immunostained for endogenous Atg16L1 (green) and endogenous EEA1 (blue). Cells were analysed as in b; the graph shows quantification of the results. Scale bar, 10 μm. Arrows indicate Atg16L1-cholera toxin co-localization; arrowheads indicate EEA1-cholera toxin co-localization. n = 20 cells.(d) HeLa cells treated with HBSS for 6 h were incubated with Alexa Fluor-555-labelled cholera toxin subunit B (red) as in a. Cells were then fixed and immunostained for endogenous Atg5 (green) or endogenous Atg12 (green). Co-localization of the Atg5 (n = 18 cells) or Atg12 vesicles (n = 39 cells) with cholera toxin is shown in yellow (arrows) and is quantified in the graph. Scale bar, 10 μm. All error bars represent s.e.m.
figf6(a) HeLa cells transfected for 24 h with Flag-tagged wild-type Atg16L1 or the Atg16L1 deletion mutants 2-275 or 232-607 Atg16L1, together with GFP-Atg5 and CFP-LC3, were fixed and immunostained with anti-Flag antibody before confocal analysis. Co-localization of wild-type or 2-275 Atg16L1 (red) with GFP-Atg5 (green) is marked by arrows in the insets. Scale bars, 10 μm.(b) HeLa cells transfected for 24 h with GFP-Atg16L1 were incubated for 15 min with horseradish peroxidase (HRP)-conjugated cholera toxin subunit B at 4 °C. The cells were then incubated for 10 min at 37 °C, after which they were processed for double immunogold labelling with anti-HRP antibodies (15-nm gold particles) and anti-GFP antibodies (10-nm gold particles) for electron microscopy. PM, plasma membrane. Scale bar, 100 nm. Co-localization can be seen in boxed areas. n = 49 fields. All error bars represent s.e.m.
figf7(a) HeLa cells transfected for 24 h with GFP-Atg16L1 (green) were incubated with CellMask Orange plasma membrane stain for 5 min at 37 °C, after which they were imaged immediately (in an incubated chamber at 37 °C). A time series after fusion of a CellMask Orange vesicle (red) with a GFP-Atg16L1 vesicle (green) is shown. Scale bar, 5 μm. A higher-magnification image showing co-localization (yellow) of GFP-Atg16L1 vesicle (green) with CellMask Orange-positive vesicle (red), indicated by arrows, is also shown in the top and bottom panels.(b, c) HeLa cells transfected with control, clathrin heavy chain, AP1 or AP2 siRNA for 72 h were subsequently transfected with GFP-Atg16L1 (green) together with the siRNA for the next 24 h. The cells were then incubated for 15 min with Alexa Fluor-555-conjugated cholera toxin subunit B (red) at 4 °C, followed by further incubation for 10 min at 37 °C, after which the cells were fixed for confocal analysis (b). The co-localization (yellow) of GFP-Atg16L1 (green) with cholera toxin (red) in control or clathrin heavy-chain knockdown is shown and is quantified in the graphs in c. n = 25 cells. Three asterisks, P = 0.0002 for clathrin heavy-chain knockdown, and P 0.0001 for AP2 knockdown. All error bars represent s.e.m. Scale bar, 5 μm.
figf8(a) HeLa cells transfected with two rounds of control, clathrin heavy chain or AP2 siRNA for 4 days were collected for immunoprecipitation (IP) with anti-Atg16L1 antibody. Western blot for total lysate (bottom) and IP (top) were performed with anti-Atg16L1 and anti-clathrin antibodies. Note the decrease in Atg16L1-clathrin interaction with AP2 knockdown. The graph shows quantification of the Atg16L1-clathrin or Atg16L1-AP2 with knockdown of control, clathrin heavy chain or AP2 from two independent experiments.(b) HeLa cells transfected for 24 h with Flag-tagged wild-type Atg16L1, GFP-PLC(PH) together with empty vector (control) or dominant-negative dynamin II mutant (K44A dynamin; DN-Dyn) were fixed and analysed by confocal microscopy. Note the co-localization of Atg16L1 with PLC(PH) at the plasma membrane; this is quantified in the graph at the right. Asterisk, P 0.01. n = 20 cells. Scale bars, 5 μm. DAPI, 4′,6-diamidino-2-phenylindole.(c) HeLa cells treated for 5 h with HBSS alone or with HBSS containing 80 μM dynasore were collected for immunoprecipitation (IP) with anti-Atg16L1 antibody. Western blot analyses for total lysate (TL) and IP were performed with anti-Atg16L1 and anti-clathrin antibodies. Note the strong Atg16L1-clathrin interaction with dynasore treatment. The graph shows quantification from two independent experiments. All error bars represent s.e.m.
f1(b) Cells stably expressing WT or D620N GFP-VPS35 or otherwise transiently transfected with a panel of GFP-VPS35 constructs were lysed and the respective GFP-tagged protein recovered by immunoprecipitation (IP).(c) Untransfected HeLa cells or cells stably expressing VPS29-GFP, GFP-VPS35 wild-type or GFP-VPS35 D620N were lysed and the lysates incubated with anti-VPS26 to IP the retromer CSC.(d) Cells stably expressing the VPS29-GFP V90D mutant that cannot assemble with VPS35 were transiently transfected with mCherry-tagged WT VPS35, D620N and H675R constructs, along with an additional control of mCherry-FAM21 tail. Cells were lysed 48 h post transfection, and VPS29-GFP V90D and associated proteins were recovered by anti-GFP native IP. Blots shown are representative of experiments replicated at least twice.
f2a) Cells expressing either WT GFP-VPS35 or the D620N mutant were treated with siRNA to abolish expression of endogenous VPS35. Each dish of cells was lysed in either HEPES lysis buffer (H) or PBS lysis buffer (P). Following centrifugation, equal portions of each lysate were combined to generate a mixed lysate (H/P). Each lysate was incubated with anti-GFP to recover the respective GFP-tagged VPS35 protein.(b) Similar to (a), but only cells expressing GFP-VPS35 wild type were analysed, either mock treated or treated with siRNA to silence FKBP15 expression.(c) Cells stably expressing WT or D620N GFP-VPS35 were treated with indicated siRNAs, lysed and immunoprecipitated as in (a).
f3(a,b) Cells expressing either WT or D620N GFP-VPS35 were treated with siRNA to silence expression of endogenous VPS35, fixed and labelled for GFP and FKBP15 (a) or FAM21 (b). Cells were imaged by fluorescence microscopy. Scale bar, 20 μm.(c) Cells stably expressing WT or D620N GFP-tagged VPS35 were treated with siRNA to abolish expression of endogenous VPS35 and subsequently fixed and labelled for GFP and either VPS26, FKBP15 or FAM21. The cells were imaged using Cellomics automated fluorescence microscopy. Approximately 250 cells per well in four wells were analysed for each cell line. The data for FKBP15 and FAM21 spot intensity were normalized to GFP-VPS35 and VPS26 signals. P0.0002 for both FKBP15 and FAM21 spot intensity in D620N compared with WT. Error bars indicate s.d.(d) Cells stably expressing either WT GFP-VPS35 or the D620N mutant were treated with siRNA to silence the expression of endogenous VPS35. Cells were permeabilized by flash freezing followed by rapid thawing and centrifuged to separate supernatant (S) and membrane pellet (P) fractions.(e) The graph shows the percentage of membrane-associated FKBP15 and strumpellin and is the mean of three experiments. The error bars indicate s.e.m. A representative blot of three experiments is shown, indicating the efficacy of the knockdown of endogenous VPS35 and also further showing that membrane proteins such as the CIMPR are detected only in the pellet fraction.
f4a) HeLa cells stably expressing GFP-VPS35 WT and D620N were treated with bafilomycin A1 or DMSO vehicle control. Endogenous LC3-II and tubulin levels were examined by western blot. A representative experiment of six experiments is shown. (b) Quantification of the representative experiment in triplicate shown in a, in which endogenous LC3-II levels are normalized to tubulin and expressed as a ratio of levels in WT. ***P=7.86 × 10−6 (DMSO) and 3.81 × 10−5 (Baf) by 2-tailed Student's t-test.c) HeLa cells stably expressing GFP-VPS35 WT and D620N were transfected with mRFP-LC3. The number of LC3 vesicles was quantified by Cellomics automated fluorescence microscopy. A representative experiment of three independent experiments is shown, with 344 (WT) and 401 (D620N) cells analysed. ***P0.0001 by 2-tailed Student's t-test.(d) GFP-VPS35 WT and D620N-expressing cells were transfected with HA-Q74 and immunostained for HA. The percentage of transfected cells with aggregates was counted by a blinded experimenter. The quantification shows the mean of three experiments in triplicate with minimum 200 cells per replicate. ***P=0.00086 by 1-tailed Student's t-test.(e) Confocal images representative of the experiment described in d. Scale bar, 20 μm.(f) Cells stably expressing WT and D620N GFP-VPS35 were transfected with GFP-α-synuclein A53T and GFP for 48 h and analyzed by western blotting.g) Quantification of the representative experiment in triplicate in f, in which the level of α-synuclein was expressed as a ratio to GFP. A representative experiment of two independent experiments is shown. **P=0.0047 by 2-tailed Student's t-test.(h) VPS35 was knocked down with two individual siRNA nucleotides in HeLa cells, and cells were treated with bafilomycin A1 and lysed as in a. A representative experiment is shown in triplicate(i) Quantification of three independent experiments in triplicate. *P=0.026; other results non-significant by 2-tailed Student's t-test.(j) Protein levels of CSC, including VPS35, VPS26 and VPS29), were assessed upon VPS35 knockdown, confirming previous results that knockdown of one component destabilizes the CSC. All error bars indicate s.e.m.
f5(a) HeLa cells were transfected with pEGFP vector or GFP-FAM21 tail for 48 h and subsequently treated with bafilomycin A1 as in Fig. 4. Endogenous LC3-II and tubulin levels were assessed by western blot. (b) Quantification of the representative experiment in triplicate shown in a, of two independent experiments. *P=0.02 (DMSO) and 0.08 (Baf) by 2-tailed Student's t-test.(c) HeLa cells stably expressing GFP-VPS35 WT and D620N were transfected with pEGFP vector or GFP-FAM21, and subsequently treated with bafilomycin A1, lysed and subjected to western blot as in a. (d) Quantification of the representative experiment in triplicate shown in c, of two independent experiments. **P=0.008 by 2-tailed Student's t-test.(e) Cells were depleted of WASH1, treated with bafilomycin A1 and examined for endogenous LC3-II, tubulin, WASH1 and GAPDH levels. A representative experiment of six independent experiments is shown. (f) Quantification of the representative experiment shown in e. **P=0.002 (DMSO) and P=0.0028 (Baf) by 2-tailed Student's t-test.g) HeLa cells depleted of WASH1 by siRNA were transfected with GFP-LC3 for 24 h. The number of LC3 vesicles was quantified by Cellomics automated fluorescence microscopy. A representative experiment of two independent experiments is shown, with 757 (control) and 695 (knockdown) cells analysed. ***P0.0001 by 2-tailed Student's t-test.(h) HeLa cells depleted of FKBP15 were transfected with GFP-LC3 and vesicles were quantified as in g. A representative experiment of two independent experiments in triplicate is shown, with 579 (control) and 815 (knockdown) cells analysed in total.(i) HeLa cells depleted of FKBP15 were subsequently transfected with a GFP-Q74 construct for 24 h and fixed in PFA. The percentage of transfected cells with aggregates was counted by a blinded experimenter. The quantification shows the mean of two experiments, each in triplicate with min 220 cells counted per replicate(j) FKBP15 was depleted in HeLa cells as in e. A representative blot from three independent experiments in triplicate is shown. Error bars indicate s.e.m.