EMBO/soda-nel huggingface dataset:
The Long Noncoding RNA ROCKI Regulates Inflammatory Gene Expression (2019)
Figure 3

Figure 3. lnc-MARCKS Negatively Regulates MARCKS Expression and promotes inflammatory responses

(A-D) Repression of lnc-MARCKS using CRISPRi increased MARCKS expression and decreased IL-6 induction. (A) Illustration of CRISPRi system and the relative locations of the two sgRNAs. (B-D) lnc-MARCKS (B), MARCKS (C), and IL-6 (D) expressions were quantified in cells stably expressing dCas9/KRAB/BFP and the NC or sgRNAs. Mean ± SD of n = 3. **p < 0.01, ***p < 0.005, ****p < 0.0001 by Student's t test.

(E) The lnc-MARCKS gene locus. The full-length sequence of lnc-MARCKS was obtained by rapid amplification of cDNA ends (RACE). lnc-MARCKS is located on human chromosome 6 and encompasses 2 exons.

(F and G) Overexpression of lnc-MARCKS decreases MARCKS expression and enhances IL-6 expression. RT-qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector (E.V.) or lnc-MARCKS, treated with PMA overnight, and left unstimulated or stimulated with Pam3 for 8 h. Lnc-MARCKS and MARCKS expression (F), IL-6 expression (G). Mean ± SD of n = 3. ***p < 0.005 by Student's t test.

(H-J) lnc-MARCKS regulates MARCKS and IL-6 in primary macrophages. Primary macrophages were transfected with NC or lnc-MARCKS antisense oligo to knock down lnc-MARCKS expression. 48 h later, cells were stimulated with Pam3 for 8 h and harvested to detect lnc-MARCKS (H), MARCKS (I), and IL-6 expressions (J). Mean ± SD of n = 3. *p < 0.05, **p < 0.01 by Student's t test.
The Long Noncoding RNA ROCKI Regulates Inflammatory Gene Expression (2019)
Figure 4

Figure 4. ROCKI (lnc-MARCKS) Binds to the MARCKS Promoter and Interacts with APEX1

(A) ROCKI is recruited to the MARCKS promoter in Pam3-stimulated THP1-derived macrophages. ChIRP analysis of THP1-derived macrophages treated with Pam3 for 8 h. ChIRP assays were performed using a non-specific lacZ probe or probes specific for lnc-MARCKS. Specificity of ROCKI probes (left), and enrichment at the MARCKS promoter (right). Probes are listed in Table EV2. Mean ± SD of n = 3. **p < 0.01, ***p < 0.005 by Student's t test.

(B) Mass spectrometry identifies APEX1 as a candidate ROCKI-binding protein in THP1-derived macrophages. APEX1 MS/MS count represents the ratio between the number of APEX1 peptides and all peptides detected. Mean of n = 2 samples. ROCKI and ROCKI-AS represent ROCKI and antisense strand of ROCKI transcripts, respectively.

(C) ROCKI inhibition of MARCKS expression is dependent on APEX1. RT-qPCR analysis of MARCKS, ROCKI, and APEX1 expression in THP1-derived macrophages overexpressing ROCKI and/or depleted of APEX1. Mean ± SD of n = 3. ***p < 0.001 by Student's t test.

(D) APEX1 specially binds to ROCKI. Western blot analysis of APEX1 in lacZ, or ROCKI pull-down fractions. Vimentin, which non-specifically binds to all RNA probes, is shown as a loading control.

(E) ROCKI associates with APEX1. Left: RT-qPCR analysis of ROCKI present in anti-APEX1 antibody or control IgG immunoprecipitates from nuclear lysates of Pam3-treated THP1-derived macrophages. Right: APEX1 western blot of the same immunoprecipitates. Mean ± SD of n = 4. ****p < 0.0001 by Student's t test.
The Long Noncoding RNA ROCKI Regulates Inflammatory Gene Expression (2019)
Figure 5

Figure 5. ROCKI Reduces H3K27ac Modification of the MARCKS Promoter via APEX1 Binding and Recruitment of HDAC1.

ChIP analysis of THP1-derived macrophages demonstrates APEX1 recruitment to the MARCKS promoter. RT-qPCR analysis of fragments of the MARCKS promoter region (a and b) or a non-specific region (nc) co-immunoprecipitated with anti-APEX1 antibody. Mean ± SD of n = 4. ****p < 0.0001 by Student's t test.

ChIP analysis of APEX1 recruitment to the MARCKS promoter was performed as described for A except the cells expressed control shRNA (NC) or ROCKI shRNA and treated with Pam3 for 8 h. The y-axis shows the percentage of enrichment in normalized to input. An unrelated region of the ACTB2 promoter was amplified as a control. Mean ± SD of n = 4. **p < 0.01. ****p < 0.0001, ns, non-significant by Student's t test.

Left: Western blot analysis of APEX1 and HDAC1 in control rabbit IgG or anti-APEX1 antibody immunoprecipitates from NC (control) or ROCKI knockdown THP1-derived macrophages. Right: RT-qPCR analysis of ROCKI in the same immunoprecipitates.

Knockdown of ROCKI decreased HDAC1 enrichment on the MARCKS promoter. ChIP-qPCR analysis of HDAC1 was performed as described for (A) using an anti- HDAC1 antibody. Mean ± SD of n = 3. *p < 0.05, ****p < 0.0001, ns, non-significant by Student's t test.

Knockdown of ROCKI enhances H3K27ac deposition on the MARCKS promoter. ChIP-qPCR analysis of H3K27ac was performed as described for (A) using an anti-H3K27ac antibody. Mean ± SD of n = 3. ****p < 0.0001, ns, non-significant by Student's t test.
The Long Noncoding RNA ROCKI Regulates Inflammatory Gene Expression (2019)
Figure 6

Figure 6. ROCKI and MARCKS are Ca2+ Regulators in TLR1/2 Signaling

Identification of genes regulated by both ROCKI and MARCKS by RNA-seq of ROCKI-overexpressing or MARCKS-knockdown THP1-derived macrophages stimulated with Pam3 for 8 h.

Heatmap of representative genes commonly upregulated in ROCKI-overexpressing and MARCKS-knockdown THP1-derived macrophages.

RT-qPCR validation of inflammatory gene expression in ROCKI-overexpressing and MARCKS-knockdown THP1-derived macrophages stimulated with Pam3 for 8 h. E.V., empty vector; NC, control shRNA. Mean ± SD of n = 4. *p < 0.05, **p < 0.01, ***p < 0.001 by Student's t test.

MARCKS is a negative regulator of Ca2+ signaling. Fluo-4 fluorescence of MARCKS, ROCKI, or NC (control) shRNA-expressing THP1-derived macrophages before and after treatment with PMA and Pam3. Triton X-100 was added to obtain the maximal [Ca2+] signal. Fluorescence levels are expressed as the change in signal normalized to the maximal fluorescence (ΔF/Fmax).
The Long Noncoding RNA ROCKI Regulates Inflammatory Gene Expression (2019)
Figure 7

Figure 7. Genetic Link Between ROCKI and Human Disease and a Model for ROCKI Function

(A) QTL signals for ROCKI (LINC01268) gene expression in whole blood (top, black), H3K27ac promoter signal in monocytes (middle, green), and H3K4me1 signal in monocytes (bottom, orange) in a 400 kb window around the MARCKS/ROCKI locus. The signals were strongly co-localized (Pshared=.97, Pshared=.98) suggesting they represent the same signal.

(B) Signed test statistics from UK Biobank association data for 1463 disease- or medication-related traits for rs9387181. Black crosses denote the mean ± SEM for each group. Dotted horizontal line indicates the null.

(C) RT-qPCR analysis of ROCKI expression in dendritic cell samples from uninfected and M. tuberculosis-infected donors. Mean ± SD of n = 6. ***p < 0.001 by Student's t test.

(D) A cis-regulatory model for ROCKI function in the negative regulation of MARCKS in innate immune cells. Upon TLR activation, ROCKI binds to the MARCKS promoter region and recruits APEX1, which facilitates HDAC1-mediated removal of the H3K27ac modification, thereby inhibiting MARCKS expression.

Extended Viewed Figures
FAM134B oligomerization drives endoplasmic reticulum membrane scission for ER-phagy (2019)
Figure 1

Figure 1. FAM134B forms oligomers that are required for ER membrane scission and ER-phagy

(A) Endogenous FAM134B forms oligomers. FAM134B knockout (KO) or wild type (WT) 293T cells were treated with 1μM of Thapsigargin (Tg) for different time and the cell lysates were prepared and analyzed by Western-blot.

(B) Analysis the oligomer size of recombinant FAM134B WT using Native PAGE.

(C) In vitro liposome fragmentation assay. After the injection of recombinant proteins (100μg/100μl) into the chamber (500μl), the morphological changes of liposomes were monitored by live imaging for 20 min. The images at different time points as indicated are presented. Scale bars, 10 μm. "ns" means no significance, one-way ANOVA, error bars indicate SEM (n=3).

(D) Quantification of the time between protein addition and liposome fragmentation. ***P<0.001, one-way ANOVA, error bars indicate SEM (n=3).

(E, F) Measurement of the intracellular ER-scission activity. U2OS cells transiently expressing EGFP-FAM134B (WT), EGFP-FAM134B (84-233) or EGFP-FAM134B (Δ84-233) at same levels, lysosomal degradation of EGFP-FAM134B was blocked by Bafilomycin A1 (Baf A1) or DMSO. GFP-positive puncta were quantified for each cell in (F). For Each group, at least 30 cells were counted. Scale bars, 10 μm. ***P<0.001, "ns" means no significance, one-way ANOVA, error bars indicate SEM.

(G, H) Measurement of the ER-phagy activity. U2OS cells transiently expressing mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (84-233) or mCherry-EGFP-FAM134B (Δ84-233) at same levels. ER-phagy is indicated by mCherry-positive but EGFP-negative puncta were quantified for each cell in (H). For mCherry-EGFP-FAM134B (WT), 50 cells were counted (n=50); for mCherry-EGFP-FAM134B (84-233), n=56; for mCherry-EGFP-FAM134B (Δ84-233), n=66. Scale bars, 10 μm. The scale bars in the magnification boxes are 2μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.

(I) Lysosomal cleavage of GFP was analyzed by Western blot for the cells co-expressing GFP-SEC61B and FAM134B WT-Flag, FAM134B (84-233)-Flag or FAM134B (Δ84-233)-Flag.
FAM134B oligomerization drives endoplasmic reticulum membrane scission for ER-phagy (2019)
Figure 2

Figure 2. Phosphorylation enhances FAM134B oligomerization and its activity in membrane scission and ER-phagy.

(A) Mass spectrometry analysis uncovered that FAM134B was phosphorylated at serine 151.

(B) Comparison of the self-interaction of FAM134B mono-phosphorylation mutants using co-IP.

(C) Comparison of the self-interaction of FAM134 tri-phosphorylation mutants using co-IP.

(D) Comparison of membrane scission activity using in vitro liposome fragmentation assay. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM (n≧3).

(E) Lysosomal cleavage of GFP was analyzed by Western blot for the cells co-expressing GFP-SEC61B and FAM134B (WT)-Flag, FAM134B (SA)-Flag or FAM134B (SD)-Flag.

(F, G) Measurement of the intracellular ER-scission activity. FAM134B knockout (KO) U2OS cells was engineered to express EGFP-FAM134B (WT), EGFP-FAM134B (SA) or EGFP-FAM134B (SD) at endogenous levels, lysosomal degradation of EGFP-FAM134B was blocked by Bafilomycin A1 (Baf A1). GFP-positive puncta were quantified for each cell in (G). For control, 25 cells were counted (n=25); For EGFP-FAM134B (WT), n=27; for EGFP-FAM134B (SA), n=29; for EGFP-FAM134B (SD), n=25. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.

(H, I) Measurement of the ER-phagy activity. FAM134B knockout (KO) U2OS cells was engineered to express mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (S151A) or mCherry-EGFP-FAM134B (S151D) at endogenous levels. Lysosomal mCherry-positive but GFP-negative puncta were quantified for each cell in (I). For mCherry-EGFP-FAM134B (WT), 25 cells were counted (n=25); for mCherry-EGFP-FAM134B (SA), n=23; for mCherry-EGFP-FAM134B (SD), n=21. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.
FAM134B oligomerization drives endoplasmic reticulum membrane scission for ER-phagy (2019)
Figure 3

Figure 3. CAMK2B phosphorylates FAM134B at Ser151 to enhance ER fragmentation and ER-phagy

(A, B) Endogenous CAMK2B redistribution to ER membrane structures labeled by mCherry-FAM134B and BAP31 upon Tg treatment. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. The colocalization was analyzed by Pearson's correlation coefficient (PCC) in (B). For Ctrl, 16 cells were counted(n=16); for Tg treatment, 18 cells were counted (n=18). ***P<0.001, one-way ANOVA, error bars indicate SEM.

(C) In vitro FAM134B S151 phosphorylation by CAMK2B was detected by Western blot. Recombinant proteins for FAM134BWT and FAM134BS151A were purified from E. coil were incubated with purified CAMK2B by IP in kinase buffer. FAM134B phosphorylation was analyzed by a specific antibody recognizing phos-Serine151 of human FAM134B.

(D) FAM134B S151 phosphorylation in cells treated with CAMK2 activator (100nM EB1089 for 1h) or/and inhibitor (10μM KN93 for 2h). 293T or SKN-SH (a cell line derived from neuroblastoma) cells were treated with drugs as indicated, whole cell lysates were analyzed by phospho-FAM134B (S151) antibody.

(E) FAM134B S151 phosphorylation in CAMK2B knockdown 293T cells.

(F) In vitro reconstitution of CAMK2B-FAM134B-mediated membrane fragmentation using liposome assay. Purified recombinant FAM134B and CAMK2B were preincubated in kinase buffer with ADP or ATP at 30°C for 10min, the resultant protein mixtures were transferred to chamber coated with liposomes. Liposome fragmentation was monitored by live imaging. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM (n=3).

(G, H) Measurement of the ER-phagy activity. U2OS cells transiently expressing mCherry-EGFP-FAM134B (WT) were treated with compounds as indicated. Lysosomal mCherry-positive but GFP-negative puncta were quantified for each cell in (H). For vehicle control, 53 cells were counted (n=53), for Ionomycin, n=56; for EB1089, n=54; for KN-93, n=58. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.

(I) FAM134B WT or knockout (KO) U2OS cells were treated with CAMK2 activator Ionomycin (Iono), EB1089 or inhibitor KN93 for 24 hours, Western-blot was performed to analyze the proteins as indicated.

(J) CAMK2B WT or knockdown (KD) U2OS cells were treated with 1 μM Thapsigargin (Tg) to induce ER stress for different time as indicated. Cells were collected and analyzed for proteins as indicated by Western blot.

(K) GFP-cleavage assay. CAMK2B WT or knockdown (KD) U2OS cells were co-transfected with FAM134B-Flag and GFP-SEC61B, 24 hours post transfection, cells were treated with drugs as indicated and analyzed for GFP-cleavage by Western blot. Note: the levels of Flag-FAM134B, CAMK2B, GFP-SEC61B and Tubulin were analyzed by Western blot using different membranes.
FAM134B oligomerization drives endoplasmic reticulum membrane scission for ER-phagy (2019)
Figure 4

Figure 4. A HSAN II patient-derived variant, FAM134BG216R is a gain-of-function mutant in oligomerization, ER scission and ER-phagy

(A) Comparison of the self-interaction of FAM134B WT with mutants as indicated using co-IP. SA-G216R was constructed by simultaneously mutating S149, S151 and S153 to alanine (SA) on the basis of FAM134B-G216R.

(B) In vitro liposome fragmentation assay. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM (n≧3).

(C, D) Measurement of the intracellular ER-scission activity of FAM134B WT, G216R, S151A-G216R and SA-G216R. GFP-tagged FAM134B proteins were expressed in U2OS cells at same levels. The autolyosomal degradation of ER fragments labelled by GFP-FAM134B were blocked by Baf A1 or DMSO. GFP-positive puncta were quantified for each cell in (D). For all of the groups, at least 30 cells were included for quantification; Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.

(E, F) Measurement of the ER-phagy activity of FAM134BG216R. U2OS cells transiently expressing mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (G216R), mCherry-EGFP-FAM134B (S151A-G216R) or mCherry-EGFP-FAM134B (SA-G216R) at same levels. Lysosomal mCherry-positive but GFP-negative puncta were quantified for each cell in (F). For WT, 50 cells were counted (n=50); for G216R, n=55; for S151A-G216R, n=51; for SA-G216R, n=52. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. *P<0.05, ***P<0.001, one-way ANOVA, error bars indicate SEM.
FAM134B oligomerization drives endoplasmic reticulum membrane scission for ER-phagy (2019)
Figure 5

Figure 5. FAM134BG216R induced sensory neuron death.

(A,A') Specific expression of FAM134B mRNA (green) in sensory neurons (red) of P4d vGlut2Cre;R26lsl-tdTommouse. Scale bars, 100μm. The white dotted line indicates the dorsal root ganglia (DRG) where sensory neurons are genetically labeled with Tomato. Insets of (a) and (a') are high magnifications of sensory neurons showing the co-localization of FAM134B mRNA and Tomato. Scale bars, 10 μm.

(B)-(G) Cultured sensory neurons from DRGs of E14.5 vGlut2Cre;R26lsl-tdTom embryos were infected with same titers of recombinant lentivirus expressing GFP-FAM134B WT (B), G216R(C), S151A-G216R(D), SA-G216R(E), G216R-ΔLIR (F) and G216R supplemented with 5μM KN93 (G), respectively. Infected sensory neurons (GFP+/Tom+) are indicated with arrowheads. The nuclei of all cells are shown with DAPI staining. Scale bars, 20 μm.

(H) Quantification of the viability of sensory neuron after 48h, 72h and 96h of infection. Sensory neuron viability was calculated as the percentage of transfected sensory neuron (GFP+/Tom+) in total sensory neurons (Tom+). For each time point, four biological replicates were included for statistical analysis. *P<0.05, **P<0.01, ***P<0.001, two-way ANOVA, error bars indicate SEM. "ns" is the abbreviation for "not significant". For all of the groups, at least 10 fields of the slides were included.
HIF2α Activation and Mitochondrial Deficit due to Iron Chelation Cause Retinal Atrophy (2022)
Figure 3

Figure 3. DFO disrupts iRPE integrity in vitro.

iRPE cells were treated with DFO at 100 mM and characterized in vitro.

(A - D) Light microscopic examination of the iRPE cells 24 and 48 hours post treatment. Scale bar: 50 µm.

(E - H) iRPE cells were stained with ZO-1 24 and 48 hours post treatment. Untreated iRPE cells were used as the control. Scale bar: 30 µm.

(I - L) Cell death among the iRPE population was examined by TUNEL assay 24 hours and 48 hours post treatment. Untreated iRPE cells were included as the control. The cells were counterstained with Hoechst. Scale bar: 30 µm.

(M) Cell viability was determined with the iRPE cells subject to DFO treatment for 24 and 48 hours. The colorimetric values of the treatment groups were normalized to the control group. The statistics are analyzed by one-way ANOVA with the Tukey test. The results are presented as mean ± S.E.M., n = 5 iRPE lines for each group. * p < 0.05; ** p < 0.01.

(N) iRPE cells were collected and lysed for immunoblotting assay against PARP, which is predicted to be 100 kDa. The molecular weight of cleaved PARP was 89 kDa.
HIF2α Activation and Mitochondrial Deficit due to Iron Chelation Cause Retinal Atrophy (2022)
Figure 4

Figure 4. DFO upregulates HIF2α and perturbs its downstream targets.

(A) iRPE cells treated with DFO were collected and lysed to measure the expression of HIF1α and HIF2α 24 and 48 hours after the treatment. The iRPE cells supplemented with 0.1% DMSO in the culture media were used as the control. Actin was used as the loading control. HIF1α (post-translationally modified) was predicted to be ~100 kDa; HIF2α (post translationally modified) was predicted to be ~ 120 kDa.

(B) iRPE cells treated with DFO were lysed and fractionated by centrifugation to measure the expression of HIF1α and HIF2α in the nuclei 24 and 48 hours after the treatment. Untreated iRPE cells were used as the normal control. Histone H3 was used as the loading control.

(C - G) The transcripts of HIFα-regulated genes were determined by qPCR with iRPE cells subject to DFO treatment for 24 hours as the template. The cDNA transcript extracted from untreated iRPE was included as the control. The expression of each transcript was normalized to ACTB as the housekeeping gene. (C) Measurement of the HIF1α and HIF2α transcripts. The statistics are analyzed by one-way ANOVA with the Tukey test. The results are presented as mean ± S.E.M., n = 3 iRPE lines for each group. *** p < 0.001. (D) Measurement of cell-survival-related transcripts regulated by HIFα. (E) Measurement of apoptosis-related transcript regulated by HIFα. (F) Measurement of glycolysis-related transcript regulated by HIFα. (G) Measurement of iron-transport-related transcript regulated by HIFα.

Data information: (D - G) The statistics are analyzed by ratio paired student's t-test. The results are presented as mean ± S.E.M., n = 3 iRPE lines for each group. * p < 0.05; ** p < 0.01; *** p < 0.001. Round dots: untreated iRPE; square dots: DFO-treated iRPE.
HIF2α Activation and Mitochondrial Deficit due to Iron Chelation Cause Retinal Atrophy (2022)
Figure 7

Figure 7. AKG preserves mitochondrial capacity and inhibits HIF2α upregulation in iRPE cells.

(A) Mitochondrial respiration of the DFO-treated (100 mM) iRPE cells with AKG supplementation (10 mM) for 24 hours was determined by seahorse extracellular flux assay. OCR was measured over a course of time. Each dot is shown as mean ± S.E.M., n = 5 iRPE lines for each group. OCR: oxygen consumption rate; FCCP: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; Rot: rotenone; AA: antimycin A.

(B, C) Maximal (B) and spare capacity (C) of mitochondrial respiration were determined based on the OCR tracing readout. The statistics are analyzed by paired student's t-test. The results are presented as mean ± S.E.M., n = 5 iRPE lines for each group. * p < 0.05.

(D) Glycolytic stress of the DFO-treated (100 mM) iRPE cells with AKG supplementation (10 mM) for 24 hours was determined by seahorse extracellular flux assay. ECAR was measured over a course of time. Each dot is shown as mean ± S.E.M., n = 5 iRPE lines for each group. ECAR: extracellular acidification rate.

(E, F) The basal (E) and the ratio (F) between mitochondrial OCR and glycolysis were determined based on the ECAR tracing readout. The statistics are analyzed by paired student's t-test. The results are presented as mean ± S.E.M., n = 5 iRPE lines for each group.

(G) The DFO-treated (100 mM) iRPE cells supplemented with AKG (10 mM) for 48 hours were lysed for immunoblotting against HIF2α. The iRPE cells supplemented with 0.1% DMSO in the culture media were used as the controls. HIF2α (post translationally modified) was predicted to be ~ 120 kDa. Actin was included as the loading control.

(H) RPE lysate from the one-year-old mice subject to 10-month DFO treatment with or without concomitant supplementation of AKG was used for immunoblotting against HIF2α. The RPE of the age-matched untreated mice was included as the control. HIF2α (degraded) was predicted to be 40 - 80 kDa. Actin was used as the loading control.
APEX2-mediated RAB proximity labeling identifies a role for RAB21 in clathrin-independent cargo sorting (2018)
Figure 1

Figure 1: APEX2:RAB expression lead to endosomal biotinylation. (A) Diagram representing APEX2:RAB-mediated endosomal biotinylation of endogenous proteins. (B) Illustrative representation of APEX2:RAB endosomal microdomains. APEX2 only or APEX2:RAB were found to biotinylate endogenous proteins. (C) Streptavidin Western blotting of total biotinylated proteins in APEX2:RAB or APEX2 only Flp-In/T-REx HeLa cells. (D) APEX2:RAB biotinylated proteins (streptavidin) are partially colocalized with EEA1 in HeLa cells, n=2 independent experiments. Scale bars: 10µm or 5µm in enlarged views.