Journal: bioRxiv

Article Title: Rabaptin5 targets autophagy to damaged endosomes and SCVs by interaction with FIP200 and ATG16L1

doi: 10.1101/2020.09.01.277764

Figure Lengend Snippet:

Article Snippet: Mouse anti-transferrin receptor (OKT8 mouse monoclonal hybridoma, kind gift of Dr. H. Farhan, 1:1000), mouse anti-Rbpt5 (610676, BD Transduction Laboratories, 1:1000) and rabbit anti-Rabaptin5 antibodies (NBP1-47285, Novus Biologicals, 1:500) were used for immunofluorescence and Western blotting, rabbit anti-FIP200 (12436S, Cell Signaling, 1:1000) was used for Western blot and co-immunoprecipitation.

Techniques: Two Hybrid Screening

Journal: bioRxiv

Article Title: Rabaptin5 targets autophagy to damaged endosomes and SCVs by interaction with FIP200 and ATG16L1

doi: 10.1101/2020.09.01.277764

Figure Lengend Snippet: A : Schematic representation of the sequence of Rabaptin5. Coiled-coil (CC) segments are shown in yellow. Colored backgrounds highlight the segments shown to interact with Rab4, Rab5, Rabex5, and the GAE and GAT domains of GGAs (Golgi-localizing, γ-adaptin ear homology domain, ARF-binding proteins). Below, the segments used to test yeast two-hybrid interaction with residues 257–444 of FIP200 are shown with their number (#) and the observed interaction (+ or –). B : Yeast two-hybrid analysis for interaction between the above-shown Rabaptin5 segments (Rbpt5#, fused to LexA on the bait plasmid) and residues 257–444 of FIP200 (FIP, fused to the Gal4 activation domain on the prey plasmid) to drive HIS3 expression. Three different clones each were replica-plated on medium with His or without His, but containing 3-amino-1,2,4-triazole (3AT; an inhibitor of His synthesis to increase stringency) and grown in the absence of Trp and leucine as a control. As negative controls, empty bait or prey plasmids were used. The asterisk indicates a clone invalidated by recombination. C : Schematic representation of the sequence of FIP200 with its coiled coil segments in yellow. Residues 281– 439 (gray) indicates the minimal sequence identified to interact with Rabaptin5 in the yeast two-hybrid screen. D : FIP200 was immunoprecipitated (IP) from lysates of HeLa or HEK293A cells and probed for FIP200, Rabaptin5 (Rbpt5), and EEA1 (early endosome antigen 1) by immunoblotting. Input lysate (10%) was immunoblotted blotted parallel. As a negative control, the immunoprecipitation was performed using an anti-GAPDH antibody. E–G : Lysates of HeLa cells transiently transfected with full-length FIP200-mCherry (FIP200-mCh) or a deletion mutant lacking the segment interacting with Rabaptin5 (Δ280–440) were immuprecipitated with anti-mCherry (IP FIP200-mCh) or, as a control, with anti-FLAG antibodies (IP FLAG). Immunoprecipitates and input lysates (10%) were immunoblotted for mCherry and Rabaptin5 (E), ATG13 (F), or ULK1 (G).

Article Snippet: Mouse anti-transferrin receptor (OKT8 mouse monoclonal hybridoma, kind gift of Dr. H. Farhan, 1:1000), mouse anti-Rbpt5 (610676, BD Transduction Laboratories, 1:1000) and rabbit anti-Rabaptin5 antibodies (NBP1-47285, Novus Biologicals, 1:500) were used for immunofluorescence and Western blotting, rabbit anti-FIP200 (12436S, Cell Signaling, 1:1000) was used for Western blot and co-immunoprecipitation.

Techniques: Sequencing, Binding Assay, Plasmid Preparation, Activation Assay, Expressing, Clone Assay, Control, Two Hybrid Screening, Immunoprecipitation, Western Blot, Negative Control, Transfection, Mutagenesis

Journal: bioRxiv

Article Title: Rabaptin5 targets autophagy to damaged endosomes and SCVs by interaction with FIP200 and ATG16L1

doi: 10.1101/2020.09.01.277764

Figure Lengend Snippet: HeLa cells were transfected with FLAG-tagged FIP200 alone (A) or together with Rabaptin5 (Rbpt5-wt) (B), GFP-Rab4 (C), RFP-Rab5 (D), or Citrine-Rab7 (E), fixed after 24 h, and subjected to immunofluorescence microscopy. Bar, 10 µm.

Article Snippet: Mouse anti-transferrin receptor (OKT8 mouse monoclonal hybridoma, kind gift of Dr. H. Farhan, 1:1000), mouse anti-Rbpt5 (610676, BD Transduction Laboratories, 1:1000) and rabbit anti-Rabaptin5 antibodies (NBP1-47285, Novus Biologicals, 1:500) were used for immunofluorescence and Western blotting, rabbit anti-FIP200 (12436S, Cell Signaling, 1:1000) was used for Western blot and co-immunoprecipitation.

Techniques: Transfection, Immunofluorescence, Microscopy

Journal: bioRxiv

Article Title: Rabaptin5 targets autophagy to damaged endosomes and SCVs by interaction with FIP200 and ATG16L1

doi: 10.1101/2020.09.01.277764

Figure Lengend Snippet: A–H : To more easily visualize Rabaptin5, a stable HEK293A cell line overexpressing Rabaptin5 (HEK +Rbpt5 ) was generated. Rabaptin5 levels were anayzed by immunoblotting in comparison to parental HEK293A cells (panel A’ ). HEK +Rbpt5 cells, untransfected or 24 h after transfection with mCherry-Galectin3, mCherry-FIP200, or mCherry-ATG16L1, without (–CQ) or with chloroquine treatment (60 µM) for 30 min (+CQ), were analyzed by immunofluorescence microscopy for Rabaptin5 and either transferrin receptor (TfR) ( A and B ), mCherry-Galectin3 ( C ), ubiquitin ( D ), mCherry-FIP200 ( E ), WIPI2 ( E ), mCherry-ATG16L1 ( G ), or LC3B ( H ). Bar, 10 µm. In the enlarged insets, arrowheads point out chloroquine-induced enlarged (circular) early endosomes. I–K : HEK +Rbpt5 cells, untransfected or 24 h after transfection with mCherry-ATG16L1, were treated with 60 µM chloroquine for 0, 15 and 30 min and stained for Rabaptin5 and either WIPI2, mCherry-ATG16L1, or LC3B. Manders’ colocalization coefficients were determined, M1 showing the fraction of Rabaptin5-positive structures also positive for WIPI2 ( I ), mCherry-ATG16L1 ( J ), or LC3B ( H ), and M2 showing the respective inverse (mean and standard deviation of three independent experiments; ANOVA: *p < 0.05, **p < 0.01, ***p<0.001).

Article Snippet: Mouse anti-transferrin receptor (OKT8 mouse monoclonal hybridoma, kind gift of Dr. H. Farhan, 1:1000), mouse anti-Rbpt5 (610676, BD Transduction Laboratories, 1:1000) and rabbit anti-Rabaptin5 antibodies (NBP1-47285, Novus Biologicals, 1:500) were used for immunofluorescence and Western blotting, rabbit anti-FIP200 (12436S, Cell Signaling, 1:1000) was used for Western blot and co-immunoprecipitation.

Techniques: Generated, Western Blot, Comparison, Transfection, Immunofluorescence, Microscopy, Ubiquitin Proteomics, Staining, Standard Deviation

Journal: bioRxiv

Article Title: Rabaptin5 targets autophagy to damaged endosomes and SCVs by interaction with FIP200 and ATG16L1

doi: 10.1101/2020.09.01.277764

Figure Lengend Snippet: A : HEK +Rbpt5 cells were treated without (–) or with 60 µM chloroquine (+CQ) or 1 mM LLOMe (+LLOMe) for 30 min and analyzed by immunofluorescence microscopy for Rabaptin5 and ALIX. B : HeLa cells stably expressing CHMP4B-GFP were treated as in panel A and analyzed for Rabaptin5, CHMP4B-GFP, and ALIX.

Article Snippet: Mouse anti-transferrin receptor (OKT8 mouse monoclonal hybridoma, kind gift of Dr. H. Farhan, 1:1000), mouse anti-Rbpt5 (610676, BD Transduction Laboratories, 1:1000) and rabbit anti-Rabaptin5 antibodies (NBP1-47285, Novus Biologicals, 1:500) were used for immunofluorescence and Western blotting, rabbit anti-FIP200 (12436S, Cell Signaling, 1:1000) was used for Western blot and co-immunoprecipitation.

Techniques: Immunofluorescence, Microscopy, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: Rabaptin5 targets autophagy to damaged endosomes and SCVs by interaction with FIP200 and ATG16L1

doi: 10.1101/2020.09.01.277764

Figure Lengend Snippet: A : HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing Rabaptin5 (siRbpt5) or FIP200 (siFIP200) for 72 h and treated without (–) or with 60 µM chloroquine (+CQ) or 250 nM Torin1 for 150 min. Below, the efficiency of Rabaptin5 and FIP200 knockdown was assayed by immunoblotting using tubulin (Tub) as a loading control. B and C : WIPI2 (B) or LC3B puncta per cell (C) were quantified for each condition (mean and standard deviation of three independent experiments; ANOVA: *p < 0.05, **p < 0.01). D : HEK293A cells were transfected with siCtr or siRbpt5 as in A and incubated without or with 280 µM LLOMe for 150 min to induce lysophagy. Cells were fixed and immunostained for endogenous WIPI2 and LC3B. Bar, 10 µm. E : WIPI2 or LC3B puncta per cell were quantified (mean and standard deviation of three independent experiments).

Article Snippet: Mouse anti-transferrin receptor (OKT8 mouse monoclonal hybridoma, kind gift of Dr. H. Farhan, 1:1000), mouse anti-Rbpt5 (610676, BD Transduction Laboratories, 1:1000) and rabbit anti-Rabaptin5 antibodies (NBP1-47285, Novus Biologicals, 1:500) were used for immunofluorescence and Western blotting, rabbit anti-FIP200 (12436S, Cell Signaling, 1:1000) was used for Western blot and co-immunoprecipitation.

Techniques: Transfection, Knockdown, Western Blot, Control, Standard Deviation, Incubation

Journal: bioRxiv

Article Title: Rabaptin5 targets autophagy to damaged endosomes and SCVs by interaction with FIP200 and ATG16L1

doi: 10.1101/2020.09.01.277764

Figure Lengend Snippet: A : HEK +Rbpt5 cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing WIPI2 (siWIPI2) for 72 h and with mCherry-ATG16L1 for 24 h. Cells were treated without or with 60 µM chloroquine (+CQ) for 30 min and stained for Rabaptin5 and mCherry-ATG16L1. Fluorescence micrographs of chloroquine-treated cells are shown (left panel). Bar, 10 µm. Arrowheads point out chloroquine-induced enlarged early endosomes. The efficiency of WIPI2 knockdown was assayed by immunoblotting using actin as a loading control (middle panel). Manders’ colocalization coefficients were determined (right panel), M1 showing the fraction of Rabaptin5-positive structures also positive for mCherry-ATG16L1 and M2 showing the inverse. Mean and standard deviation of three independent experiments; ANOVA: *p < 0.05, **p < 0.01. B : Parental HEK293A and FIP200 knockout cells (FIP200-KO) were transfected with mCherry-ATG16L1 for 24 h and incubated without or with 60 µM chloroquine (+CQ) for 30 min and stained for Rabaptin5 and mCherry-ATG16L1. Fluorescence micrographs of chloroquine-treated cells are shown (left panel). Bar, 10 µm. Arrowheads point out chloroquine-induced enlarged early endosomes. Manders’ colocalization coefficients were determined (right panel). Mean and standard deviation of five independent experiments; ANOVA: *p < 0.05.

Article Snippet: Mouse anti-transferrin receptor (OKT8 mouse monoclonal hybridoma, kind gift of Dr. H. Farhan, 1:1000), mouse anti-Rbpt5 (610676, BD Transduction Laboratories, 1:1000) and rabbit anti-Rabaptin5 antibodies (NBP1-47285, Novus Biologicals, 1:500) were used for immunofluorescence and Western blotting, rabbit anti-FIP200 (12436S, Cell Signaling, 1:1000) was used for Western blot and co-immunoprecipitation.

Techniques: Transfection, Staining, Fluorescence, Knockdown, Western Blot, Control, Standard Deviation, Knock-Out, Incubation

Journal: bioRxiv

Article Title: Rabaptin5 targets autophagy to damaged endosomes and SCVs by interaction with FIP200 and ATG16L1

doi: 10.1101/2020.09.01.277764

Figure Lengend Snippet: A : HeLa cells transiently transfected with full-length mCherry-ATG16L1 were treated with 60 µM chloroquine for 0, 30, or 120 min, lysed, and immunoprecipitated with anti-Rabaptin5 (IP: Rbpt5) or, as a control, with anti-FLAG antibodies (IP FLAG). Immunoprecipitates and input lysates (10%) were immunoblotted for Rabaptin5 and ATG16L1. Signals were quantified and the ratios of mCherry-ATG16L1/Rabaptin5 normalized to that without (0 min) chloroquine treatment (mean and standard deviation of three independent experiments; ANOVA: *p < 0.05). B : Co-immunoprecipitation was performed as in panel A using parental HEK293A cells and CRISPR/Cas9 knockout cells lacking FIP200 (FIP-KO). Anti-HA antibodies were used as a control (IP HA). On the right, HEK293A and FIP200 knockout cells were immunoblotted for FIP200 and as a loading control of tubulin (Tub). C : Lysates of HEK293A or HeLa cells transiently transfected with full-length mCherry-ATG16L1 (wt) or a mutant lacking the WD domain (ΔWD) were immunoprecipitated with anti-Rabaptin5 or anti-FLAG antibodies, and immunoblotted for Rabaptin5 and ATG16L1. D : The consensus sequence of the ATG16L1 interaction motifs of TMEM59, NOD2, and TLR2 (above; ) is shown together with the matching sequence in Rabaptin5 (below). The three point mutations to alanine to produce the AAA mutant of Rabaptin5 are indicated. E : Lysates of HeLa cells transiently transfected with myc-tagged wild-type Rabaptin5 (wt) or triple-alanine mutant (AAA) were immunoprecipitated with anti-myc (IP myc) or anti-FLAG antibodies (IP FLAG), and immunoblotted for myc and ATG16L1.

Article Snippet: Mouse anti-transferrin receptor (OKT8 mouse monoclonal hybridoma, kind gift of Dr. H. Farhan, 1:1000), mouse anti-Rbpt5 (610676, BD Transduction Laboratories, 1:1000) and rabbit anti-Rabaptin5 antibodies (NBP1-47285, Novus Biologicals, 1:500) were used for immunofluorescence and Western blotting, rabbit anti-FIP200 (12436S, Cell Signaling, 1:1000) was used for Western blot and co-immunoprecipitation.

Techniques: Transfection, Immunoprecipitation, Control, Standard Deviation, CRISPR, Knock-Out, Mutagenesis, Sequencing

Journal: bioRxiv

Article Title: Rabaptin5 targets autophagy to damaged endosomes and SCVs by interaction with FIP200 and ATG16L1

doi: 10.1101/2020.09.01.277764

Figure Lengend Snippet: A : By immunoblot analysis, the levels of Rabaptin5 and as a loading control of tubulin (Tub) were assessed in wild-type HEK293A cells, HEK +Rbpt5 cells stably overexpressing Rabaptin5, and Rabaptin5-knockout cells without (Rbpt5-KO) or with stable re-expression of wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA). B : The same stable HEK293A-derived cell lines were treated without (–CQ) or with 60 µM chloroquine for 150 min (+CQ) and analyzed by immunofluorescence microscopy for WIPI2 or LC3B. C : WIPI2 and LC3B puncta per cell were quantified for each condition (mean and standard deviation of three independent experiments; ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). D and E: The HEK293A-derived cell lines were treated with 60 µM chloroquine for 30min and non-lipidated and lipidated LC3B (I and II, resp.) were assayed by immunoblot analysis (D). The increase of the LC3B-II fraction of total LC3B upon chloroquine treatment was quantified (mean and standard deviation of three independent experiments; ANOVA: *p < 0.05, **p < 0.01). F : HEK +Rbpt5 cells and Rabaptin5-knockout cells stably re-expressing wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA) were transfected with mCherry-ATG16L1, treated with 60 µM chloroquine (CQ) for 0, 15 and 30 min, and analyzed by immunofluorescence microscopy for Rabaptin5 and mCherry-ATG16L1. Manders’ colocalization coefficients were determined, M1 showing the fraction of Rabaptin5-positive structures also positive for mCherry-ATG16L1 and M2 showing the inverse (mean and standard deviation of three independent experiments; ANOVA for Rbpt5-KO+wt or +AAA vs. HEK +Rbpt5 cells: *p < 0.05, **p < 0.01, ***p < 0.001). G : The same three cell lines as in panel F, but untransfected, were analyzed in the same way for Rabaptin5 and WIPI2. H : Wild-type HEK293A cells, HEK +Rbpt5 cells, Rabaptin5-knockout cells without (Rbpt5-KO) or with stable re-expression of wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA) were stained with lysotracker and with DAPI for nuclei. Bar, 10 µm. I : The number of lysosomes (lysotracker-positive structures) per cell was quantified from cells as in panel C (mean and standard deviation of three independent experiments). Images for at least 15 cells per sample were quantified. The value for HEK +Rbpt5 cells is an underestimate, because the density of puncta makes them difficult to distinguish as separate structures. ANOVA: *p < 0.05, ****p < 0.0001.

Article Snippet: Mouse anti-transferrin receptor (OKT8 mouse monoclonal hybridoma, kind gift of Dr. H. Farhan, 1:1000), mouse anti-Rbpt5 (610676, BD Transduction Laboratories, 1:1000) and rabbit anti-Rabaptin5 antibodies (NBP1-47285, Novus Biologicals, 1:500) were used for immunofluorescence and Western blotting, rabbit anti-FIP200 (12436S, Cell Signaling, 1:1000) was used for Western blot and co-immunoprecipitation.

Techniques: Western Blot, Control, Stable Transfection, Knock-Out, Expressing, Mutagenesis, Derivative Assay, Immunofluorescence, Microscopy, Standard Deviation, Transfection, Staining

Journal: bioRxiv

Article Title: Rabaptin5 targets autophagy to damaged endosomes and SCVs by interaction with FIP200 and ATG16L1

doi: 10.1101/2020.09.01.277764

Figure Lengend Snippet: A: Treatment of HEK293A and Rabaptin5-KO cells with 60 µM chloroquine for 30 min similarly leads to swelling of early endosomes positive for EEA1 as detected by immunofluorescence. B: HEK293A cells and Rabaptin5-KO cells were transduced to express tandem fluorescent RFP-GFP-LC3, starved in HBSS or treated with 250 nM Torin1 for 150 min, fixed and subjected to fluorescnet microscopy. Images for at least 16 cells per sample were quantified. C: The number of autophagosomes (GFP+/RFP+) and autolysosomes (GFP /RFP+) LC3 puncta were counted and autophagosomes plotted as a percentage of the total (mean and standard deviation of three independent experiments).

Article Snippet: Mouse anti-transferrin receptor (OKT8 mouse monoclonal hybridoma, kind gift of Dr. H. Farhan, 1:1000), mouse anti-Rbpt5 (610676, BD Transduction Laboratories, 1:1000) and rabbit anti-Rabaptin5 antibodies (NBP1-47285, Novus Biologicals, 1:500) were used for immunofluorescence and Western blotting, rabbit anti-FIP200 (12436S, Cell Signaling, 1:1000) was used for Western blot and co-immunoprecipitation.

Techniques: Immunofluorescence, Microscopy, Standard Deviation

Journal: bioRxiv

Article Title: Rabaptin5 targets autophagy to damaged endosomes and SCVs by interaction with FIP200 and ATG16L1

doi: 10.1101/2020.09.01.277764

Figure Lengend Snippet: A : HEK +Rbpt5 cells, 24 h after transfection with mCherry-ATG16L1, were treated with 100 µM monensin for 0, 15 and 30 min and stained for Rabaptin5,WIPI2, and mCherry-ATG16L1 to assess their colocalization on swollen early endosomes. Bar, 10 µm. B and C : Manders’ colocalization coefficients were determined, showing the fraction of Rabaptin5-positive structures also positive for mCherry-ATG16L1 (B) or for WIPI2 (C) (mean and standard deviation of three independent experiments, quantifying ∼40 cells for each sample; ANOVA for Rbpt5-KO+AAA vs. HEK +Rbpt5 cells: *p < 0.05, **p < 0.01). D and E : Wild-type HEK293A cells, HEK +Rbpt5 cells, and Rabaptin5-knockout cells without (Rbpt5-KO) or with stable re-expression of wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA) were treated without (–Mon) or with 100 µM monensin for 150 min (+Mon), and analyzed by immunofluorescence microscopy for WIPI2 or LC3B. WIPI2 (D) of LC3B (E) puncta per cell were quantified for each condition (mean and standard deviation of four independent experiments; ANOVA: *p < 0.05, **p < 0.01, ****p < 0.0001).

Article Snippet: Mouse anti-transferrin receptor (OKT8 mouse monoclonal hybridoma, kind gift of Dr. H. Farhan, 1:1000), mouse anti-Rbpt5 (610676, BD Transduction Laboratories, 1:1000) and rabbit anti-Rabaptin5 antibodies (NBP1-47285, Novus Biologicals, 1:500) were used for immunofluorescence and Western blotting, rabbit anti-FIP200 (12436S, Cell Signaling, 1:1000) was used for Western blot and co-immunoprecipitation.

Techniques: Transfection, Staining, Standard Deviation, Knock-Out, Expressing, Mutagenesis, Immunofluorescence, Microscopy

Journal: bioRxiv

Article Title: Rabaptin5 targets autophagy to damaged endosomes and SCVs by interaction with FIP200 and ATG16L1

doi: 10.1101/2020.09.01.277764

Figure Lengend Snippet: A : HeLa cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing Rabaptin5 (siRbpt5) or FIP200 (siFIP200) for 72 h. The cells were infected with Salmonella by centrifugation at 500×g for 5min at 37°C and incubation for 10 min at 37°C, washed three times, and incubated in fresh culture medium containing gentamicin to prevent growth of extracellular bacteria for 0, 1, 3, or 6 h before lysis of the host cells and plating of the bacteria on LB agar plates at various dilutions to determine the number of live bacteria at the different time points, shown as a percentage of internalized cells after infection (mean and standard deviation of three independent experiments). On the right, the fractions of internalized bacteria alive 1 h after infection are shown separately (mean and standard deviation of three independent experiments; ANOVA: *p < 0.05). B : Wild-type HEK293A, HEK +Rbpt5 , Rbpt5-KO, Rbpt5-KO+wt, Rbpt5-KO+AAA, and FIP200-KO cells were infected with Salmonella and treated and analyzed as in panels A (mean and standard deviation of three independent experiments; ANOVA: *p < 0.05, ****p < 0.0001). C : Wild-type HEK293A, HEK +Rbpt5 , Rbpt5-KO, Rbpt5-KO+AAA, and FIP200-KO cells were infected with Salmonella expressing GFP as in panel B, incubated in fresh culture medium containing gentamicin for 0, 5, 15, 30, and 60 min, fixed with methanol and immunostained for transferrin receptor (TfR) as a marker of early endosomes and for LC3B as a marker of autophagy. Salmonella were classified according to their association with a TfR- and/or LC3B-positive compartment – as illustrated on the top left (bar, 2 µm) – during the first hour after infection. In the absence of Rabaptin5, LC3-positive SCVs with early endosomal characteristics (containing TfR) were strongly reduced. (Means and standard deviations of three independent experiments, analyzing >50 bacteria for each time point.)

Article Snippet: Mouse anti-transferrin receptor (OKT8 mouse monoclonal hybridoma, kind gift of Dr. H. Farhan, 1:1000), mouse anti-Rbpt5 (610676, BD Transduction Laboratories, 1:1000) and rabbit anti-Rabaptin5 antibodies (NBP1-47285, Novus Biologicals, 1:500) were used for immunofluorescence and Western blotting, rabbit anti-FIP200 (12436S, Cell Signaling, 1:1000) was used for Western blot and co-immunoprecipitation.

Techniques: Transfection, Infection, Centrifugation, Incubation, Bacteria, Lysis, Standard Deviation, Expressing, Marker

Journal: bioRxiv

Article Title: Rabaptin5 targets autophagy to damaged endosomes and SCVs by interaction with FIP200 and ATG16L1

doi: 10.1101/2020.09.01.277764

Figure Lengend Snippet: HeLa cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing Rabaptin5 (siRbpt5) or FIP200 (siFIP200) for 72 h, infected with Salmonella expressing GFP and washed as in Figure 8A and immediately fixed for fluorescence microscopy and stained with anti-transferrin receptor and anti-LC3B. Z-stacks for >5’000cells/sample were acquired and analyzed in Fiji to determine the fraction of infected cells (mean and standard deviation of three independent experiments). The average number of bacteria per infected cell was identical (2.16, 2.11, and 2.12 bacteria per cell transfected with siCtr, siRbpt5, and siFIP200, resp.).

Article Snippet: Mouse anti-transferrin receptor (OKT8 mouse monoclonal hybridoma, kind gift of Dr. H. Farhan, 1:1000), mouse anti-Rbpt5 (610676, BD Transduction Laboratories, 1:1000) and rabbit anti-Rabaptin5 antibodies (NBP1-47285, Novus Biologicals, 1:500) were used for immunofluorescence and Western blotting, rabbit anti-FIP200 (12436S, Cell Signaling, 1:1000) was used for Western blot and co-immunoprecipitation.

Techniques: Transfection, Infection, Expressing, Fluorescence, Microscopy, Staining, Standard Deviation, Bacteria