sec61β specific antibody  (Proteintech)

Journal: Cell reports

Article Title: Legionella uses host Rab GTPases and BAP31 to create a unique ER niche

doi: 10.1016/j.celrep.2024.115053

Figure Lengend Snippet: (A) HeLa Fcγ cells were infected with L.p. for the indicated times, stained with anti-Rtn4 (sER marker), Sec61β (rER marker) antibodies, and Hoechst ( L.p .) for immunofluorescence analysis. Results represent three independent experiments (100 vacuoles were scored in each). Error bars represent mean ± SD. Bar, 5 μm. (B) HeLa Fcγ cells stably expressing GFP-Rtn4 and RFP-Sec61β were infected with Halo-tagged WT L.p . for 3 h for FLIP. The top panel represents GFP-Rtn4 before photobleaching, and the bottom represents GFP-Rtn4 after photobleaching for 80 s. Bar, 10 μm. (C) The graph represents the relative fluorescence intensity of GFP-Rtn4 (green lines) and RFP-Sec61β (red lines) in the ER and around LCV. Error bars represent mean ± SD. The purple and red boxes represent the area around LCV and bleach areas, respectively. (D) HeLa Fcγ cells stably expressing GFP-Rtn4 and RFP-Sec61β were infected with WT L.p . for 6 h. The top panel represents RFP-Sec61β before photobleaching, and the bottom represents RFP-Sec61β after photobleaching. The purple and red boxes represent the area around the LCV and bleach areas, respectively. Bar, 10 μm. (E) The graph represents the relative fluorescence intensity of GFP-Rtn4 (green lines) and RFP-Sec61β (red lines) in the ER and around LCV. (F) HeLa Fcγ cells stably expressing GFP-Rtn4 and RFP-Sec61β were infected for 6 h, and RFP-Sec61β was photobleached for FRAP. The green box represents the total area of bleach. The red and blue boxes represent the bleached area around the LCV and the ER, respectively. White arrows represent the area around LCV. Bar, 10 μm. (G) The graph represents the relative fluorescence intensity of GFP-Rtn4 (green lines) and RFP-Sec61β (red lines) in the ER and around LCV. Error bars represent mean ± SD.

Article Snippet: Anti-Sec61β rabbit polyclonal antibody , Proteintech , Cat# 14846-1-AP.

Techniques: Infection, Staining, Marker, Immunofluorescence, Stable Transfection, Expressing, Fluorescence

Journal: Cell reports

Article Title: Legionella uses host Rab GTPases and BAP31 to create a unique ER niche

doi: 10.1016/j.celrep.2024.115053

Figure Lengend Snippet: (A) HeLa Fcγ cells were subjected to subcellular fractionation, and immunoblot analysis was done against the indicated proteins. (B) Sucrose gradient fractionation was done to obtain the sER and rER fractions and immunoblotted against indicated antibodies. (C) HeLa Fcγ cells expressing mRFP-Rab4 or mRFP-Rab10 were infected with L.p. for the indicated times, stained with L.p . for immunofluorescence analysis, and the vacuoles positive for mRFP-Rab4 or -Rab10 were quantified. Bar, 5 μm. The purple box represents the magnified image. Results represent three independent experiments (100 vacuoles were scored in each). Error bars represent mean ± SD. (D) HEK293 Fcγ cells were transfected with either mock, Rab4, or Rab10 siRNA for 72 h, and cell extracts were immunoblotted against the indicated antibodies. (E and F) HeLa Fcγ cells were silenced for Rab4 or Rab10 for 72 h, infected with L.p. for 4 and 8 h, and the vacuoles positive for Rtn4 or Sec61β were counted. Results represent three independent experiments, and statistical significance was measured using one-way ANOVA (100 vacuoles were scored in each experiment). Bars represent mean ± SD. (G) HeLa Fcγ cells were silenced with Rab4 and Rab10 siRNA, and the ER morphology was observed by immunofluorescence analysis. Bars, 5 μm. Arrows indicate disruption of the sER tubular network. Arrowheads indicate diffusion of rER to the cell periphery. (H) Triplicate experiments (30 cells for each condition) from 3G were quantitated and are shown as a histogram. Bars represent mean ± SD.

Article Snippet: Anti-Sec61β rabbit polyclonal antibody , Proteintech , Cat# 14846-1-AP.

Techniques: Fractionation, Western Blot, Expressing, Infection, Staining, Immunofluorescence, Transfection, Disruption, Diffusion-based Assay

Journal: Cell reports

Article Title: Legionella uses host Rab GTPases and BAP31 to create a unique ER niche

doi: 10.1016/j.celrep.2024.115053

Figure Lengend Snippet: (A) HeLa Fcγ cells were infected with L.p . for the indicated time, fixed, and stained with anti-BAP31 antibody and Hoechst 33342. White boxes indicate the LCV. (B) HeLa Fcγ cells were transfected with either mock or BAP31 siRNA for 72 h and immunoblotted against the indicated antibodies. (C and D) Mock-treated or BAP31-silenced HeLa Fcγ cells were infected with L.p . for the indicated time, fixed, and stained with anti-Rtn4 or -Sec61β antibodies and DAPI, and the vacuoles were quantified. Results are representative of three independent experiments (100 vacuoles were scored in each experiment). Bars represent mean ± SD. (E) HeLa Fcγ cells were silenced with either mock or BAP31 for 72 h, followed by infection with L.p. for 8 h. Post infection, cells were fixed and stained with anti-Rtn4 antibody and Hoechst for immunofluorescence analysis. Bar, 5 μm. (F) HeLa Fcγ cells were silenced with either mock or BAP31 for 72 h, followed by infection with L.p. for 12 h at an MOI of 2. Post infection, cells were fixed and stained with anti- Legionella antiserum for detection of extracellular bacteria, then permeabilized and further stained with DAPI for detection of intracellular bacteria. Bar, 5 μm. The graph shows the percentage of vacuoles containing a single bacterium, 1–10 bacteria, 11–30 bacteria, and ≥31 bacteria in a single vacuole. Data are representative of three independent experiments (30 vacuoles were scored in each experiment). Results are shown as the mean ± SD. *** p < 0.001.

Article Snippet: Anti-Sec61β rabbit polyclonal antibody , Proteintech , Cat# 14846-1-AP.

Techniques: Infection, Staining, Transfection, Immunofluorescence, Bacteria

Journal: Cell reports

Article Title: Legionella uses host Rab GTPases and BAP31 to create a unique ER niche

doi: 10.1016/j.celrep.2024.115053

Figure Lengend Snippet: (A) Schematic representation of the chromosomal organization of genomic islands of L.p . (B) HeLa Fcγ cells were infected with the indicated genomic island deletion mutant L.p. strains for 4 h and processed for immunofluorescence against BAP31 antibody. The histogram represents the percentage of vacuoles positive for BAP31 post infection with different L.p. strains. (C) HeLa Fcγ cells were transfected with 3xFLAG-Lpg1152 plasmid and processed for immunofluorescence microscopy using FLAG (green) and BAP31 (red) antibodies. Nuclei were stained with DAPI. Bar, 10μm. (D and E) HEK293 Fcγ cells were transfected with the indicated plasmids. Cell extracts were subjected to immunoprecipitation assay post transfection with anti-FLAG antibody and immunoblotted against the indicated antibodies. (F) The docked model complex of Lpg1152 (deep salmon) and BAP31 (green). (G) The electrostatic potential surface of Lpg1152 is shown bound with BAP31 (green). (H) Interacting residues of Lpg1152 (orange, ball-stick) and BAP31 (lime, ball-stick) at the protein-protein interface. (I) HeLa Fcγ cells were infected with the indicated L.p. strains. Histograms represent the infection efficiency, i.e., the percentage of infected cells at MOI 10 1.5 h after infection ( n = 200 per triplicate experiment). (J) HeLa Fcγ cells were infected with the indicated strains of L.p. for the indicated time points. Following infection, LCV positive for BAP31 was scored using immunofluorescence analysis. (K) HeLa Fcγ cells were infected with the indicated strain, and the Rtn4- and Sec61β-positive vacuoles were scored using immunofluorescence. Graphs represent the percentage recruitment of Rtn4 and Sec61β at the indicated time points post infection. (L) HeLa Fcγ cells were infected with the indicated L.p. strains, and the number of L.p. per LCV was counted 8 h after infection ( n = 30 per triplicate experiment). (M) U937 cells were infected with the indicated L.p. strains for 1 h, and colony-forming units (CFU) were counted at the indicated time after infection. Data represent three independent experiments, and statistical significance was carried out with a Student’s t test. Bar represents mean ± SEM.

Article Snippet: Anti-Sec61β rabbit polyclonal antibody , Proteintech , Cat# 14846-1-AP.

Techniques: Infection, Mutagenesis, Immunofluorescence, Transfection, Plasmid Preparation, Microscopy, Staining, Immunoprecipitation

Journal: Cell reports

Article Title: Legionella uses host Rab GTPases and BAP31 to create a unique ER niche

doi: 10.1016/j.celrep.2024.115053

Figure Lengend Snippet: (A) HeLa Fcγ cells were silenced with mock, BAP31, or BAP29 siRNA for 72 h. Post silencing, cells were fixed and stained with anti-Rtn4 and Sec61β antibodies and were subjected to immunofluorescence imaging. Bar, 5 μm. White arrows indicate Sec61β localization to the cell periphery. (B) HeLa Fcγ cells were silenced with either mock or Stx-18 siRNA for 72 h. Post silencing, cells were fixed and stained with anti-Rtn4 and anti-Sec61β antibodies. Bar, 5 μm. (C and D) HeLa Fcγ cells were silenced for Stx18 alone or a combination of Stx18/BAP31 or Stx18/BAP29 siRNA. Following single or double silencing, cell extracts were prepared and immunoblotted against the indicated antibodies. (E) HeLa Fcγ cells were silenced for Stx18 alone, Stx18/BAP31, or Stx18/BAP29; following single or double silencing, cells were fixed and stained with an anti-Rtn4 antibody for immunofluorescence analysis. Bar, 5 μm.

Article Snippet: Anti-Sec61β rabbit polyclonal antibody , Proteintech , Cat# 14846-1-AP.

Techniques: Staining, Immunofluorescence, Imaging

Journal: Cell reports

Article Title: Legionella uses host Rab GTPases and BAP31 to create a unique ER niche

doi: 10.1016/j.celrep.2024.115053

Figure Lengend Snippet:

Article Snippet: Anti-Sec61β rabbit polyclonal antibody , Proteintech , Cat# 14846-1-AP.

Techniques: Virus, Recombinant, Transfection, In Situ, Fluorescence, Stable Transfection, Expressing, Software

Journal: The Journal of Biological Chemistry

Article Title: The Translocon Sec61? Localized in the Inner Nuclear Membrane Transports Membrane-embedded EGF Receptor to the Nucleus *

doi: 10.1074/jbc.M110.158659

Figure Lengend Snippet: EGFR associates with the translocon Sec61β in the INM. A, A431 cells maintained in a serum-starved medium for 24 h were treated with EGF followed by cellular fractionation. B, proteins in A were immunoprecipitated (IP) using anti-EGFR followed by immunoblotting. C, INM portions had undetectable cross-contamination with the process of cellular fractionation. MDA-MB-468 cells maintained in a serum-starved medium for 24 h were treated with EGF in a different period followed by cellular fractionation as described in the legend for Fig. 2 and subjected to immunoblotting with the indicated antibodies. The relative density of the INM-EGFR immunoblotting at zero time was defined as 1 after subtraction of the background by using the ImageJ software program (version 1.38x) to quantify the signals. The EGFR blotting of the left panel has five times shorter exposure than that of right panel. D, EGFR associated with Sec61β in the INM portions but not the NP portions in response to EGF in a time-dependent manner. The purified INM and NP portions in C were immunoprecipitated using the indicated antibodies (Abs.) followed by immunoblotting. E, co-localization of EGFR and Sec61β in the INM was analyzed using immuno-EM. An ultrathin section of MDA-MB-468 cells treated with EGF was immunostained with EGFR (goat anti-mouse IgG, 1-nm gold particles, arrows) and Sec61β (goat anti-rabbit IgG, 10-nm gold particles, arrowheads). Bar, 1 μm. PM, plasma membrane; Cy, cytoplasm.

Article Snippet: The gold particles labeling Sec61β were confirmed by two different specific primary anti-Sec61β antibodies obtained from Upstate Biotech Millipore ( supplemental Fig. S5, upper inset panel , arrows ) and from Proteintech ( supplemental Fig. S5, lower panel , arrows ), which demonstrated that the localization of Sec61β was primarily detectable in the INM.

Techniques: Cell Fractionation, Immunoprecipitation, Western Blot, Software, Purification, Clinical Proteomics, Membrane

Journal: The Journal of Biological Chemistry

Article Title: The Translocon Sec61? Localized in the Inner Nuclear Membrane Transports Membrane-embedded EGF Receptor to the Nucleus *

doi: 10.1074/jbc.M110.158659

Figure Lengend Snippet: Association of EGFR with Sec61β in the nucleus assists INM-anchored EGFR in releasing to the nucleus. A, knockdown of Sec61β prevented EGF-dependent transport of EGFR from the INM to the NP in HeLa cells. Cells were transfected with an siRNA targeting Sec61β (siRNA-Sec61β-3) (+) or a nonspecific control siRNA (−) using electroporation. Proteins from the total lysates, INM, and NP by cellular fractionation were then analyzed using immunoblotting with the antibodies as indicated. Emerin and Sp1 were used as markers for the INM and NP portions, respectively. B, knockdown of Sec61β by two individual siRNAs targeting Sec61β (siRNA-Sec61β-1 and siRNA-Sec61β-2) in HeLa cells up-regulated EGF-dependent EGFR translocation to the INM. Cells were transfected with two individual siRNAs targeting Sec61β (siRNA-Sec61β-1 and siRNA-Sec61β-2) or a nonspecific control siRNA (−) using electroporation. C, exogenous Sec61β rescued the effect of Sec61β knockdown on INM-anchored EGFR. Cells were co-transfected with a Sec61β siRNA targeting its 3′-untranslated region (UTR) (siRNA-Sec61β-3) and a 3′-UTR-deleted FLAG-tagged Sec61β using electroporation. The bar diagram indicates the relative densities of the immunoblots as quantified using the ImageJ software program (version 1.38x). The relative density by quantification is plotted diagrammatically as shown. Similar results were obtained in 2–4 four independent experiments.

Article Snippet: The gold particles labeling Sec61β were confirmed by two different specific primary anti-Sec61β antibodies obtained from Upstate Biotech Millipore ( supplemental Fig. S5, upper inset panel , arrows ) and from Proteintech ( supplemental Fig. S5, lower panel , arrows ), which demonstrated that the localization of Sec61β was primarily detectable in the INM.

Techniques: Knockdown, Transfection, Control, Electroporation, Cell Fractionation, Western Blot, Translocation Assay, Software