Journal: bioRxiv

Article Title: The involvement of the ER-phagy receptor FAM134B in membrane contact sites between ER and endolysosomes promotes ERLAD

doi: 10.1101/2025.07.08.663682

Figure Lengend Snippet: ( A ) CLSM analysis showing GFP-VAPA, V5-ORP1L and LAMP1 distribution in HALO-ATZ transfected CRISPR STX17 MEFs treated with 50nM BafA1. ( B ) Same as A for cells expressing HALO-NHK. ( C ) WB of the total cell extract (TCE) of HEK293 cells transfected with empty vector (lane 1), FAM134B-V5 (lane 2), ATZ-HA (lane 3), FAM134B-V5 and ATZ-HA (lane 4), or FAM134B-V5 and NHK-HA (lane 5), incubated for 6 h with 100 nM BafA1 and then lysed with 2% CHAPS. Membranes were probed with anti-V5, anti-CNX, anti VAPA, anti-ORP1L, anti-Rab7, anti-HA and anti-GAPDH antibodies, respectively ( D ) WB of the anti-V5 immunocomplexes of C. Membranes were probed with anti-V5, anti-CNX, anti-VAPA, anti-ORP1L, anti-Rab7 and anti-HA antibodies, respectively. ( E ) Quantification of the co-immunoprecipitation of CNX with FAM134B observed in D , the association in mock-transfected cells is set to 1. One-way ANOVA and Dunnett’s multiple comparisons test, * P < 0.1, ** P < 0.01. F=23.06. n=4 independent experiments for pCDNA3 and ATZ, n=3 independent experiments for NHK. ( F ) Quantification of the co-immunoprecipitation of VAPA with FAM134B observed in D . One-way ANOVA and Dunnett’s multiple comparisons test, ns P > 0.05, ** P < 0.01. F=13.00. n=4 independent experiments ( G ) Quantification of the co-immunoprecipitation of ORP1L with FAM134B observed in D . One-way ANOVA and Dunnett’s multiple comparisons test, ns P > 0.05, *** P < 0.001. F=26.58. n=4 independent experiments ( H ) Quantification of the co-immunoprecipitation of RAB7 with FAM134B observed in D . One-way ANOVA and Dunnett’s multiple comparisons test, ns P > 0.05, * P < 0.1. n=3 independent experiments F=5.540. Scale bar: 10μm.

Article Snippet: Commercial antibodies used in the study were rabbit HA (Sigma), rat HA (Novus), V5 (Thermo Fisher), GAPDH (Thermo Fisher), VAPA (Bioss), VAPB (Abcam), ORP1L (Abcam), RAB7 (Abcam), FLAG (Sigma), LAMP1 clone 1D4B (Hybridoma Bank, 1D4B was deposited to the DSHB by J.T.

Techniques: Transfection, CRISPR, Expressing, Plasmid Preparation, Incubation, Immunoprecipitation

Journal: bioRxiv

Article Title: The involvement of the ER-phagy receptor FAM134B in membrane contact sites between ER and endolysosomes promotes ERLAD

doi: 10.1101/2025.07.08.663682

Figure Lengend Snippet: ( A ) WB analysis showing the knockout of ORP1L in HeLa cells. ( B ) CLSM analysis of ATZ-HA delivery to LAMP1-positive endolysosomes in HeLa WT cells treated with 100nM BafA1. ( C ) CLSM analysis of ATZ-HA delivery to LAMP1-positive endolysosomes in HeLa CRISPR ORP1L cells mock transfected, treated with 100nM BafA1. ( D ) CLSM analysis of ATZ-HA delivery to LAMP1-positive endolysosomes in HeLa CRISPR ORP1L cells transfected with GFP-ORP1L, treated with 100nM BafA1. ( E ) CLSM analysis of HALO-ATZ delivery to LAMP1-positive endolysosomes in HeLa CRISPR ORP1L cells transfected with FLAG-Rab7, treated with 100nM BafA1. ( F ) CLSM analysis of HALO-ATZ delivery to LAMP1-positive endolysosomes in HeLa CRISPR ORP1L cells transfected with GFP-ORP1L and FLAG-Rab7, treated with 100nM BafA1. ( G ) CLSM analysis of ATZ-HA delivery to LAMP1-positive endolysosomes in HeLa CRISPR ORP1L cells transfected with CHERRY-ORP1L FY/AA and FLAG-Rab7, treated with 100nM BafA1. ( H ) Quantification of B-G (n = 14, 12, 8, 19, 24 and 22 cells of 3 independent experiments, respectively). One-way ANOVA and Dunnett’s multiple comparisons test, ns P > 0.05, ** P < 0.01, **** P < 0.0001. F=56.76. Scale bar: 10μm.

Article Snippet: Commercial antibodies used in the study were rabbit HA (Sigma), rat HA (Novus), V5 (Thermo Fisher), GAPDH (Thermo Fisher), VAPA (Bioss), VAPB (Abcam), ORP1L (Abcam), RAB7 (Abcam), FLAG (Sigma), LAMP1 clone 1D4B (Hybridoma Bank, 1D4B was deposited to the DSHB by J.T.

Techniques: Knock-Out, CRISPR, Transfection

Journal: bioRxiv

Article Title: The involvement of the ER-phagy receptor FAM134B in membrane contact sites between ER and endolysosomes promotes ERLAD

doi: 10.1101/2025.07.08.663682

Figure Lengend Snippet: Luminal expression of ATZ polymers and persistent cycles of de-glucosylation by α-glucosidase II (αGLCII) and re-glucosylation by UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1) of the oligosaccharide at position 83 of the ATZ polypeptide prolong the association of ATZ polymers with the lectin chaperone calnexin (CNX) . This leads to the formation of a segregation complex between CNX and the ER-phagy receptor FAM134B and, as shown here, stabilizes ER-endolysosome contacts involving the FAM134B interactor VAPA, the endolysosome-associated small GTPase RAB7 and the connecting protein ORP1L. Membrane fusion events controlled by the SNARE proteins STX17, SNAP29 and VAMP8 allow delivery of ATZ polymers within endolysosomes for clearance .

Article Snippet: Commercial antibodies used in the study were rabbit HA (Sigma), rat HA (Novus), V5 (Thermo Fisher), GAPDH (Thermo Fisher), VAPA (Bioss), VAPB (Abcam), ORP1L (Abcam), RAB7 (Abcam), FLAG (Sigma), LAMP1 clone 1D4B (Hybridoma Bank, 1D4B was deposited to the DSHB by J.T.

Techniques: Expressing, Membrane

Journal: PLoS Pathogens

Article Title: Tenuivirus utilizes its glycoprotein as a helper component to overcome insect midgut barriers for its circulative and propagative transmission

doi: 10.1371/journal.ppat.1007655

Figure Lengend Snippet: (A and B) RSV virions were detected in the early endosomes inside midgut epithelial cells by labeling using the Rab5 antibody (A) or in the late endosomes by labeling using the Rab7 antibody (B). Localization of RSV virions in the Rab5 antibody-labeled early endosomes or in the Rab7 antibody-labeled late endosomes are indicated with arrows. Actin antibody was used to visualize actin filaments inside the midgut epithelial cells. Bar, 25 μm. (C and D) NSvc2-N and NSvc2-C were detected inside the early endosomes labeled with the EEA1 antibody. Co-localizations of NSvc2-N or NSvc2-C with EEA1 or actin in different endosomes are indicated with arrows. Bar, 25 μm. (E) RSV virions:NSvc2-N complexes were released from the actin labelled late endosomes into the cytosol of epithelial cells. The white dashed cycles indicate the regions where the RSV virions:NSvc2-N complexes were detected in the cytosol. Bar, 25 μm. (F) RSV virions was released into the cytosol of epithelial cells but not NSvc2-C. The white dashed boxes indicate a region where RSV virions were released from the endosomes while NSvc2-C was still associated with endosomes. Bar, 25 μm. (G) RSV virions were released from the Rab7 antibody-labeled late endosomes into the cytosol of epithelial cells. The released RSV virions are indicated with arrows. ML, midgut lumen; EC, epithelial cells; Bar, 25μm.

Article Snippet: Rab5 anti-rabbit IgG and Rab7 anti-rabbit IgG were from the Cell Signaling Technology (Danvers, MA, USA), and EEA1 anti-mouse IgG was from Novus (Centennial, CO, USA).

Techniques: Labeling