EMBO/soda-nel huggingface dataset:
ESCRT-mediated lysosome repair precedes lysophagy and promotes cell survival (2018)
Figure 5

Figure 5. CHMP4B is recruited before Galectin3 to the damaged endolysosomal membranes.

Representative movie montage of a live-cell imaging experiment illustrating the dynamics of CHMP4B-eGFP and mCherry-Galectin3 recruitment at different time points before and after LLOMe treatment. Using the perfusion system, 250 μM LLOMe was added to HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 and images were acquired every 20 sec for a period of 20 min. As shown in the representative movie montage and the quantification graph, CHMP4B recruitment precedes Galectin3, which is an established marker for detection of damaged endolysosomal membranes. The quantification graph is normalized to the area occupied by the cells. Data were obtained from >40 cells per condition from three independent experiments. Error bars correspond to 95% confidence intervals. Data information: Scale bars: 5 μm.
ESCRT-mediated lysosome repair precedes lysophagy and promotes cell survival (2018)
Figure 6

Figure 6. ESCRT-III recruitment to damaged lysosomes precedes lysophagy.

A. HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 were incubated with 250 μM LLOMe, fixed at different time points as indicated and stained for ubiquitin. As presented, CHMP4B is recruited before ubiquitin and Galectin3 upon lysosomal membrane damage.

B. HeLa cells stably expressing CHMP4B-eGFP were treated as in (A) and stained for p62. CHMP4B is recruited before p62 on the damaged lysosomes. The number of LAMP1-positive foci appears stable independently of LLOMe treatment.

C. Experimental setup as in (A and B) where HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 were stained for LC3. As shown, CHMP4B is recruited prior to LC3.

Data information: Quantification graphs are normalized to the cell number where error bars represent 95% confidence intervals. Data were obtained from >150 cells per condition from three independent experiments.
ESCRT-mediated lysosome repair precedes lysophagy and promotes cell survival (2018)
Figure 7

Figure 7. ESCRTs are essential for cell viability after endolysosomal damage.

A. HeLa cells were transfected with different siRNAs as indicated. 48 h post-transfection cells were treated with 250 μM LLOMe for 3, 6, 10 hours harvested and stained for Annexin V. Cells were immediately processed for flow cytometry. Already after 3 h of treatment with LLOMe, cells co-transfected with TSG101+ALIX siRNA showed an increase in apoptotic cell death, whereas TSG101 siRNA transfected cells showed elevated Annexin V staining after 6 h of treatment.

B. Experimental set up as in (A) with additional propidium iodide (PI) staining of transfected HeLa cells.

Data information: Graphs show average data from a set of three independent experiments where >10,000 cells per condition were analyzed. Error bars represent SEM. Statistical significance was determined using one-way ANOVA test. p-values are presented where significant. *p-value≤ 0.05, **p-value≤ 0.01 and ***p-value≤ 0.001.
ESCRT-mediated lysosome repair precedes lysophagy and promotes cell survival (2018)
Figure 8

Figure 8. ESCRT-III and Galectin3 are recruited to the Coxiella burnetii vacuole. A. HeLa cells stably expressing CHMP4B-eGFP were transfected with the mCherry-Galectin3 plasmid. 24 h later, cells were infected with WT Coxiella burnetti. Live-cell imaging started 24 h post-infection and several time points are indicated during the next 24 h of observation (time indicated in minutes, see Movie EV 9). Arrows indicate Coxiella-containing vacuole (DRAQ5 labeling) becoming positive for CHMP4B-eGFP and mCherry-Galectin3. Data information: Scale bars: 50 μm. B. Bacterial viability and replication assay upon TSG101 KD. HeLa cells were treated with either siCtrl or with two different siTSG101 for 48 h. Then cells were infected with mCherry-Coxiella burnetii for 48 h before lysis and serial dilutions were made to infect Vero cells. 72 h later, infected cells were fixed, DAPI stained and processed for quantitative image analysis. Between 30 and 40 fields representing more than 9,000 cells per condition of three independent experiments were analyzed. Error bars represent SD. Statistical significance was determined using one-way ANOVA test. **p-value≤ 0.01 and ***p-value≤ 0.001. Legends to Expanded View Figures
ULK1-mediated phosphorylation of ATG16L1 promotes xenophagy, but destabilises the ATG16L1 Crohn's mutant (2019)
Figure 1

Figure1

ATG16L1 is phosphorylated by ULK1

(A) in vitro kinase assays were performed using purified recombinant kinases (ULK1 and ULK2) and substrate (ATG16L1) in the presence of radiolabelled ATP. ULK and ATG16L1 inputs were examined by western blot (WB) and substrate phosphorylation was analyzed by autoradiography (AR).

(B) Full-length or truncated versions of ATG16L1 were subjected to an in vitro ULK1 kinase assay. ULK1 and ATG16L1 inputs were examined by western blot and target phosphorylation by autoradiography.

(C) ATG16L1 was phosphorylated in an in vitro ULK1 kinase reaction and analysed by mass spectrometry. Phosphorylation of S278 and S287 in human (S278 marked in green, S287 marked in grey) was identified with high and low confidence, respectively. Conservation of amino acids 254-294 are shown using the Shapely colour scheme. Mass spectrometry was performed on a single experiment.

(D) Full-length or mutated HA-ATG16L1 was purified from mammalian cells and subjected to an in vitro ULK1 kinase assay. Inputs were analysed by WB and target phosphorylation by AR.

(E) HEK293A cells were transfected with wild-type or phospho-dead ATG16L1 in the presence of wild-type or kinase-dead ULK1. Phosphorylation of ATG16L1 (S278 or S287) and inputs were examined by WB.

Data information: Unless otherwise indicated experiments were performed three times.
ULK1-mediated phosphorylation of ATG16L1 promotes xenophagy, but destabilises the ATG16L1 Crohn's mutant (2019)
Figure 2

Figure 2

ULK1/2 is required for phosphorylation of ATG16L1 and xenophagy induction

(A) Wild-type, ULK1/2 double knockout (dKO), or IKKα KO mouse embryonic fibroblasts (MEFs) were incubated with either complete medium, amino acid-deficient DMEM, or HBSS for 1 hour. Samples were immunoblotted using the indicated antibodies.

(B) Wild-type, ULK1/2 dKO, or IKKα KO MEFs were infected with log phase Salmonella for 2 hours; bacteria-containing media was then removed and cells were incubated with gentamycin (50 µg/mL)-containing DMEM for 2 hours. Samples were immunoblotted using the indicated antibodies.

(C) Wild-type, ULK1/2 dKO, or IKKα KO MEFs cells were infected with Salmonella for 1 hours. Autophagic capture of Salmonella was analyzed by immunostaining for LPS and LC3B. Representative images are shown (scale bars, 10 μm and 3 μm). Quantification was generated from 8 fields of view from a representative experiment. The experiments were repeated twice.

(D) Wild-type, ULK1/2 dKO and IKKα KO MEFs were infected with Salmonella for 1 hour. Xenophagy rates were examined through Colony Forming Unit (CFU) assays. Quantification of infection rates by immunofluorescence is demonstrated in the right panel.

Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.
ULK1-mediated phosphorylation of ATG16L1 promotes xenophagy, but destabilises the ATG16L1 Crohn's mutant (2019)
Figure 3

Figure 3

ULK1 promotes cleavage of T300A ATG16L1 through phosphorylation on S278

(A) HEK293A cells were transfected with either flag-tagged WT ATG16L1 or T300A ATG16L1. ULK1 was co-transfected in increasing amounts where indicated. Cleavage of ATG16L1 was analyzed by WB of whole cell lysates. Levels of ATG16L1 cleavage were quantified from 3 biological repeats (right panel).

(B) HEK293A cells were transfected with either tagged wild-type, T300A, or S278/T300A ATG16L1 in the presence or absence of ULK1. Cleavage of ATG16L1 was analyzed by WB. Levels of ATG16L1 cleavage were measured from 3 biological repeats (right panel).

(C) HEK293A cells were transfected with the indicated plasmids in the presence or absence of a pan-caspase inhibitor Z-VAD-FMK (15 µM) for 4 hours. Cleavage of ATG16L1 was analyzed by WB of 3 biological repeats.

(D) Wild-type or T300A-expressing HEK293A were treated with Salmonella in the presence or absence of ULK1/2 inhibitor (16 μM) for the indicated time points. Expression of ATG16L1 was analysed by WB. The experiments were performed twice.

(E) ATG16L1 knock-out HEK293A cells transfected with the indicated HA GST ATG16L1 plasmids were infected with Salmonella for 1 hour. Xenophagy rates were examined through CFU assays. Quantification of infection rates by immunofluorescence is demonstrated in the right panel.

Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.
ULK1-mediated phosphorylation of ATG16L1 promotes xenophagy, but destabilises the ATG16L1 Crohn's mutant (2019)
Figure 4

Figure 4

ULK1-mediated phosphorylation is required for ATG16L1 localization to Salmonella site and bacterial clearance

(A) Wild-type MEF cells were infected with Salmonella for 25 minutes. Phospho-ATG16L1, total ATG16L1, and LPS were stained and analysed by immunofluorescence. Representative immunofluorescent images are shown (scale bars, 10 μm and 1 μm).

(B) Wild-type and ULK1/2 dKO were infected with Salmonella for 25 minutes. Immunofluorescence was performed using antibodies against LPS and ATG16L1. Representative immunofluorescent images are shown on the left panel (scale bars, 10 μm and 2 μm). Quantification of ATG16L1-positive bacteria from 7 fields of view from a representative experiment is shown in the right panel.

(C) ATG16L1 knock-out HCT116 transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 hour. Bacteria were stained using anti-LPS antibodies to analyze localization in addition to ATG16L1. Representative immunofluorescent images of ATG16L1 and LPS are shown (scale bars, 5μm and 1 μm). Quantification of ATG16L1 localizing to bacteria from 7 fields of view from a representative experiment is shown in the lower panel.

(D) ATG16L1 knock-out HCT116 transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 hour. Bacteria were stained using anti-LPS antibodies to analyze localization in addition to the autophagy marker LC3B. Representative immunofluorescent images of LC3B and LPS are shown (scale bars, 5μm and 1 μm). Quantification of bacteria undergoing autophagic clearance from 7 fields of view from a representative experiment is shown in the lower panel.

(E) A diagram demonstrating our working model for the role of ULK1-mediated phosphorylation at S278 in wild-type and T300A ATG16L1 background.

Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.

Expanded View Figure Legends
Disrupting HIV-1 capsid formation causes cGAS sensing of viral DNA (2020)
Figure 1

Fig. 1. PI treatment induces HIV-1 to trigger an ISG response in macrophages.

(A) Immunoblot of HIV-1 GFP virus particles (2×1011 genomes) produced with lopinavir (LPV, 0-100 nM) detecting p24 antibody.

(B-C) Titration of LPV-treated HIV-1 GFP viruses on PMA-treated THP-1 shSAMHD1 (B) or U87 (C) cells. Mean ± SD, n=3.

(D-F) ISG qPCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line).

(G) CXCL-10 protein in cell supernatants from D-F (ELISA).

(H) ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with 0.2 U RT/ml 30 nM LPV-treated HIV-1 GFP in the presence of DMSO vehicle or 2 μM ruxolitinib. A control was stimulated with 1 ng/ml IFNβ.

(I) RT products from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with 0.3 U RT/ml LPV-treated HIV-1 GFP viruses.

(J) Immunoblot of HIV-1 R9 BaL virus particles (2×1011 genomes) produced with LPV (0-100 nM) detecting p24.

(K, L) ISG qRT-PCR (K) and infection data (L) from primary monocyte-derived macrophages (MDM) infected for 24 h with LPV-treated HIV-1 R9 BaL viruses (0.2 U RT/ml). Data are collated from two donors (represented by circles and squares), n=3. Horizontal line represents the median. For experiments in which the virus dose used was normalised by RT activity, the number of genome copies was also measured by qPCR of virus. This gave dose equivalents of within 2-3 fold of RT equivalents.

Data information: Data are mean ± SD, n=3, representative of 2 repeats (H, I), or 3 repeats (D-G). Statistical analyses were performed using the Student's t-test, with Welch's correction where appropriate and comparing to the 0 nM LPV condition. * P<0.05, ** P<0.01, *** P<0.001. See also Figure EV1.
Disrupting HIV-1 capsid formation causes cGAS sensing of viral DNA (2020)
Figure 2

Fig 2: HIV-1 with Gag protease cleavage mutation induces ISGs in macrophages.

(A) Immunoblot of HIV-1 GFP virus particles (2×1011 genomes) with varying proportions of ΔCA-SP1 protease cleavage mutation detecting p24.

(B) Titration of HIV-1 GFP ΔCA-SP1 viruses on U87 cells. Mean ± SD, n=3.

(C, D) ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line).

(E) CXCL-10 protein in supernatants from (C, D) (ELISA).

(F) RT products from THP-1 cells transduced for 24 h with 6×109 genomes/ml (approx. 0.5 U RT/ml) HIV-1 GFP ΔCA-SP1 viruses.

(G) IFIT-1 reporter activity from monocytic THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.016 - 0.2 U RT/ml). Data are shown as individual measurements, representative of 2 repeats.

(H) IFIT-1 reporter activity from monocytic THP-1-IFIT-1 cells transduced with HIV-1 GFP containing either 0 % (WT) or 75 % ΔCA-SP1 mutant, or stimulated with 4 μg/ml cGAMP as a control, in the presence of DMSO vehicle or 2 μM ruxolitinib.

(I) ISG qRT-PCR from primary MDM transduced for 24 h with WT HIV-1 GFP or 75 % ΔCA-SP1 mutant (3×108 genomes/ml or 1.5×109 genomes/ml, equivalent to 0.1 U RT/ml and 0.5 U RT/ml).

(J) ISG qRT-PCR from primary MDM transduced for 24 h with WT HIV-1 GFP or 75 % ΔCA-SP1 mutant (1.5×109 genomes/ml), or stimulated with 1 ng/ml IFNβ, in the presence of DMSO vehicle or 2 μM ruxolitinib.

(K) CXCL-10 protein in supernatants from (J) (ELISA).

Data information: Data are mean ± SD, n=3, representative of 2 repeats (F, I-K) or 3 repeats (C-E, H). Statistical analyses were performed using the Student's t-test, with Welch's correction where appropriate and comparing to the 0 % ΔCA-SP1 virus (C-E, I) or the DMSO control (H, J, K). * P<0.05, ** P<0.01, *** P<0.001. See also Figure EV 2.
Disrupting HIV-1 capsid formation causes cGAS sensing of viral DNA (2020)
Figure 3

Fig. 3. Innate immune activation is RT-dependent.

(A) IFIT-1 reporter activity from THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP containing 0 % (WT) or 75 % ΔCA-SP1 mutant (1 U RT/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir.

(B) CXCL-10 protein in supernatants from (A) (ELISA).

(C-D) ISG qRT-PCR from THP-1-IFIT-1 cells transduced for 24 h with 0 % (WT) or 75 % ΔCA-SP1 mutant (109 and 3×109 genomes/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir.

(E) IFIT-1 reporter activity from THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP containing 0 % ΔCA-SP1 (WT), 75 % ΔCA-SP1, 75 % ΔCA-SP1 carrying a mutation in reverse transcriptase (75 % ΔCA-SP1 RT D185E) or 75 % ΔCA-SP1 carrying a mutation in integrase (75 % ΔCA-SP1 INT D116N) (3.75×109, 7.5×109 and 1.5×1010 genomes/ml).

(F) CXCL-10 protein in supernatants from (E) (ELISA). Triangles indicate CXCL-10 not detected.

Data information: Data are mean ± SD, n=3, representative of 2 repeats. Statistical analyses were performed using the Student's t-test, with Welch's correction where appropriate. * P<0.05, ** P<0.01, *** P<0.001. See also Figure EV3.
Disrupting HIV-1 capsid formation causes cGAS sensing of viral DNA (2020)
Figure 4

Fig. 4. Innate immune activation is DNA sensing-dependent.

(A) IFIT-1 reporter activity from PMA-treated THP-1-IFIT-1 shSAMHD1 cells lacking STING or MAVS, or a gRNA control (Ctrl) cell line transduced for 24 h with HIV-1 GFP 75 % ΔCA-SP1 (0.4 U RT/ml) or stimulated by transfection with either HT-DNA (0.1 μg/ml) or poly I:C (0.5 μg/ml).

(B-C) IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line stimulated for 24 h with poly I:C (transfection, 0.5 μg/ml), HT-DNA (transfection, 0.1 μg/ml), LPS (50 ng/ml) or cGAMP (transfection, 0.5 μg/ml) (B) or transduced for 24 h with HIV-1 GFP containing either 0 % (WT) or 75 % ΔCA-SP1 (1×1010 and 2×1010 genomes/ml) (C).

(D) CXCL-10 protein in supernatants from (C) (ELISA). Triangles indicate CXCL-10 not detected.

(E) IFIT-1 reporter activity from PMA-treated THP-1-IFIT-1 shSAMHD1 cells lacking STING, MAVS or matching gRNA control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml).

(F) IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or matching control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml).

(G) CXCL-10 protein in supernatants from (E).

(H) CXCL-10 protein in supernatants from (F) (ELISA).

(I) IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 control cells transduced for 48 h with WT, DRV-treated (DRV, 12.5 nM) or HIV-1 GFP containing 90 % ΔCA-SP1 (1×1010 genomes/ml) in the presence of DMSO vehicle, 2 μM ruxolitinib or 0.5 μg/ml H151.

Data information: Data are mean ± SD, n=3, representative of 2 (E-I) or 3 (A-D) repeats. Statistical analyses were performed using the Student's t-test, with Welch's correction where appropriate. * P<0.05, ** P<0.01, *** P<0.001. See also Figure EV4.
Disrupting HIV-1 capsid formation causes cGAS sensing of viral DNA (2020)
Figure 6

Fig. 6. PF-74 treatment induces HIV-1 to trigger a DNA-sensing dependent ISG response.

(A) Infection data for THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.1-3 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM).

(B) IFIT-1 reporter activity in supernatant from (A).

(C-D) ISG qRT-PCR from monocytic THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.25-1 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM).

(E) CXCL-10 protein in supernatants of THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (3 U/ml) in the presence of DMSO vehicle or PF-74 (10 μM).

(F) IFIT-1 reporter activity from THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (3 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM) and ruxolitinib (Rux, 2 μM) as indicated.

(G) IRF reporter activity from THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line transduced for 24 h with HIV-1 GFP (3 U/ml and 6 U/ml) in the presence of DMSO vehicle or PF-74 (10 μM).

(H) IFIT-1 reporter activity from THP-1-IFIT-1 cells lacking MAVS or a matching gRNA control (Ctrl) cell line transduced for 24 h with HIV-1 GFP (3 U/ml and 6 U/ml) in the presence of DMSO vehicle or PF-74 (10 μM).

Data information: Data are mean ± SD, n=2 (C-E, G), 3 (A, F), 4 (H) or 6 (B), representative of 3 repeats. Statistical analyses were performed using the Student's t-test, with Welch's correction where appropriate. * P<0.05, ** P<0.01, *** P<0.001. See also Figure EV5.
Regulation of TORC1 by Rag GTPases in nutrient response (2008)
figf1

(a) Knockdown of dRagA and dRagC decreased dS6K phosphorylation (Thr 398). Drosophila S2 cells untreated (lane 2) or treated with the indicated RNAi were starved of amino acids for 1 h followed by amino acid stimulation for 30 min Phosphorylation and protein levels of dS6K were determined by immunoblotting with the indicated antibodies. (b) dRagA and dRagC are not required for dAkt phosphorylation. Drosophila S2 cells untreated (lanes 1–3) or treated with the indicated RNAi were starved of amino acids for 1 h and stimulated with amino acids for 30 min. Phosphorylation and protein levels of dAkt were determined by immunoblotting with appropriate antibodies as indicated. NC, negative control RNAi. (c) dRagA and dRagC function in parallel with TSC2 and PTEN. dRagA and/or dRagC RNAi was added to S2 cells in combination with dTSC2 or dPTEN RNAi, as indicated. dTSC2 or dPTEN RNAi treatment increased pdS6K (Thr 398) and this increase was compromised by dRagA or/and dRagC RNAi. Full scans of blots are provided in Supplementary Information, Fig. S6.
Regulation of TORC1 by Rag GTPases in nutrient response (2008)
figf2

(a) Constitutively active RagA and RagB stimulate S6K phosphorylation. 200 ng of each mammalian RagA, RagB, RagC or RagD construct was co-transfected with HA–S6K (20 ng) into HEK293 cells. Their corresponding dominant-negative mutants (RagAT21N, RagBT54N, RagCS75N, RagDS76N) and constitutively active mutants (RagAQ66L, RagBQ99L, RagCQ120L, RagDQ121L) were also tested. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated. (b) RagA has a dominant role over RagC in regulating S6K phosphorylation. Each Rag construct (200 ng) indicated was co-transfected with HA–S6K (20 ng). The different Rag mutants used in the transfection are indicated at the top of each lane. (c) Rag regulates TORC1 but not TORC2 activity. Each indicated Rag construct (200 ng) was co-transfected with either HA–S6K (20 ng), Myc–4EBP1 (20 ng) or of GST–Akt (100 ng). Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated. Full scans of blots are provided in Supplementary Information, Fig. S6.
Regulation of TORC1 by Rag GTPases in nutrient response (2008)
figf3

(a) RagAQ66L and RagCS75N activate TORC1 in the absence of amino acids. Each Rag construct (200 ng) indicated was co-transfected with HA–S6K (20 ng) into HEK293 cells. Cells were starved of amino acids for 1 h before collection. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated. (b) RagAT21N and RagCQ120L block S6K phosphorylation in response to amino acid (AA) stimulation. pcDNA3 (200 ng, lanes 1 and 2) or each indicated Rag construct was co-transfected with HA–S6K into HEK293 cells. Cells were starved of amino acids for 1 h (–AA) and either remained in the starvation medium or were stimulated with amino-acid-containing-medium (+AA) for 30 min before collection. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated. (c) RagAT21N and RagCQ120L suppress insulin-induced stimulation of S6K phosphorylation. pcDNA3 (200 ng, lanes 1 and 2) or each indicated Rag construct was co-transfected with HA–S6K (20 ng) or GST–Akt (100 ng) into HeLa cells. Cells were serum-starved overnight and stimulated with insulin (400 nM) for 30 min. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated. Full scans of blots are provided in Supplementary Information, Fig. S6.