hsc70 wb  (Boster Bio)

Journal: bioRxiv

Article Title: DENND6A couples Arl8b to a Rab34/RILP/dynein complex regulating retrograde lysosomal trafficking and autophagy

doi: 10.1101/2023.08.21.554162

Figure Lengend Snippet: (A) Purified GST, GST-Rab34 QL and GST-Rab34 TN were incubated with lysates from HEK-293T cells expressing DENND6A-GFP. Specifically bound proteins were detected by immunoblot with anti-GFP antibody. The starting material (SM) was run in parallel to detect the total DENND6A-GFP. (B) Quantification of experiment in A ; means ± SEM; two-tailed unpaired t test (*** P ≤ 0.0005; n = 3). (C) GFP or DENND6A-GFP expressing HEK-293T cell lysates were incubated with purified T7-RILP protein. Specifically bound proteins were detected by immunoblot with anti-Rab34 antibody. The starting material (SM) was run in parallel to detect the total Rab34. Anti-GFP was used to detect GFP or DENND6A-GFP proteins and anti-HSC70 antibody was used as a loading control. (D) Quantification of experiment in C ; means ± SEM; two-tailed unpaired t test (** P ≤ 0.0025; n = 3). (E) In vitro GEF assays using purified Rab34 with or without DENND6A as indicated. The amount of [ 35 S]GTPγS transferred to the Rab34 was determined by collecting the reactions on filters, followed by scintillation counting. The relative incorporation of [ 35 S]GTPγS is plotted over time; data represent mean ± SEM; n = 3. The curve was fitted by nonlinear regression one-phase association. (F) HeLa cells co-expressing DENND6A-GFP and mCherry-Rab34 were fixed and stained with LAMP1 antibody. The cell periphery is outlined by a white dotted line. Scale bar = 10 µm for low magnification images; 3.13 µm for high magnification images. (G) HeLa cells treated with control or Rab34 siRNA were transfected with DENND6A-GFP. 16 h post transfection, cells were fixed and stained with LAMP1 antibody. The cell periphery is outlined by a white dotted line. Scale bar = 10 µm. (H) Graphical representation of cumulative distribution of LAMP1 intensity in experiments performed in G ; mean ± SEM; extra sum of F-squares test following nonlinear regression and curve fitting; n= 30 cells from 3 replicates. (I) Immunoblot showing the Rab34 protein levels in control and Rab34 siRNA treated HeLa cells. Immunoblot probed with anti-Rab34 and anti-GAPDH antibodies. (J) WT and DENND6A KO HeLa cells were transfected with GFP-Rab34. 16 h post transfection, cells were fixed and stained with LAMP1 antibody. The cell periphery is outlined by a white dotted line. Scale bar = 10 µm. (K) Graphical representation of cumulative distribution of LAMP1 intensity in experiments performed in J ; mean ± SEM; extra sum of F-squares test following nonlinear regression and curve fitting; n= 36 and 34 cells from 3 replicates corresponding to WT and DENND6A KO cells.

Article Snippet: Rabbit polyclonal GFP (A-6455) is obtained from Invitrogen (WB-1:5,000), and rat monoclonal HSC70 antibody (WB-1:10,000) is obtained from Enzo (ADI-SPA-815-F).

Techniques: Purification, Incubation, Expressing, Western Blot, Two Tailed Test, In Vitro, Staining, Transfection

Journal: bioRxiv

Article Title: DENND6A couples Arl8b to a Rab34/RILP/dynein complex regulating retrograde lysosomal trafficking and autophagy

doi: 10.1101/2023.08.21.554162

Figure Lengend Snippet: (A) Lysates from HEK-293T cells expressing DENND6A-GFP were incubated with wither beads covalently linked to T7 antibody or T7 linked beads coupled to T7-RILP protein. Specifically bound proteins were detected by immunoblot with anti-GFP antibody, anti-dynein intermediate chain (DIC) antibody and anti-Rab34 antibody. The starting material (SM) was run in parallel to detect the total DENND6A-GFP, DIC and Rab34. (B) Immunoblot showing DIC protein levels in control and dynein siRNA treated HeLa cells. Immunoblot probed with anti-DIC and anti-HSC70 antibodies. (C) Immunoblot showing RILP protein levels in control and RILP siRNA treated HeLa cells. Immunoblot probed with anti-RILP and anti-HSC70 antibodies. (C-E) HeLa cells were treated with (C) control siRNA or (D) dynein siRNA or (E) RILP siRNA and transfected with DENND6A-GFP. 16 h post transfection, cells were fixed and stained with LAMP1 antibody. The cell periphery is outlined by a white dotted line. Scale bars = 10 μm. (F) Graphical representation of cumulative distribution of LAMP1 intensity in experiments performed in C-E ; mean ± SEM; extra sum of F-squares test following nonlinear regression and curve fitting; n = 29, 30, 30 cells from 3 replicates corresponding to control, dynein, and RILP siRNA treated cells.

Article Snippet: Rabbit polyclonal GFP (A-6455) is obtained from Invitrogen (WB-1:5,000), and rat monoclonal HSC70 antibody (WB-1:10,000) is obtained from Enzo (ADI-SPA-815-F).

Techniques: Expressing, Incubation, Western Blot, Transfection, Staining

Journal: bioRxiv

Article Title: DENND6A couples Arl8b to a Rab34/RILP/dynein complex regulating retrograde lysosomal trafficking and autophagy

doi: 10.1101/2023.08.21.554162

Figure Lengend Snippet: (A) Unstarved or Earle’s Balanced Salt Solution (EBSS) starved HeLa cells were fixed, stained with LAMP1 antibody and imaged using confocal microscopy (Leica SP8). The cell periphery is outlined by a white dotted line. Scale bar = 10 µm. Yellow arrows indicate the presence of peripheral lysosomes. (B) Graphical representation of cumulative distribution of LAMP1 intensity in experiments performed in A (under starvation condition); mean ± SEM; extra sum of F-squares test following nonlinear regression and curve fitting; n = 28, 29, 30 cells, corresponding to WT, DENND6A KO1 and DENND6A KO2. (C) Immunoblot showing LC3B-II protein levels under various conditions (unstarved; EBSS starved; and EBSS starved + Bafilomycin A1 (BafA1)) Arl8a and Arl8b protein levels in control and Arl8(a+b) siRNA treated HeLa cells. Immunoblot probed with anti-LC3B-II and anti-HSC70 antibodies. (D) Quantification of experiment in F ; means ± SEM; two-way ANOVA (** P ≤ 0.0025; *** P ≤ 0.0005; **** P ≤ 0.0001; n = 3). (E) HeLa WT and DENND6A KOs cells were fixed and stained with LC3B-II antibody and DAPI. The cell periphery is outlined by a white dotted line. Scale bar = 10 µm. (F) Quantification of experiment in E ; means ± SEM; Kruskal-Wallis test (*** P = 0.0004; n = 36, 34 and 42, corresponding to WT, DENND6A KO1 and DENND6A KO2).

Article Snippet: Rabbit polyclonal GFP (A-6455) is obtained from Invitrogen (WB-1:5,000), and rat monoclonal HSC70 antibody (WB-1:10,000) is obtained from Enzo (ADI-SPA-815-F).

Techniques: Staining, Confocal Microscopy, Western Blot

Journal: Cell Reports

Article Title: Disruption of pancreatic stellate cell myofibroblast phenotype promotes pancreatic tumor invasion

doi: 10.1016/j.celrep.2021.110227

Figure Lengend Snippet:

Article Snippet: Anti-HSC70 (WB) , Santa Cruz , Cat. No.:sc-7298; RRID: AB_627761 ; Clone: B-6.

Techniques: Transduction, Virus, Plasmid Preparation, Recombinant, In Vivo, SYBR Green Assay, In Vitro, Membrane, Stripping Membranes, Luciferase, RNA Sequencing, Gene Expression, Control, Sequencing, Expressing, Derivative Assay, Software