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sec61α (Cell Signaling Technology Inc)

Name: sec61α
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Cell Signaling Technology Inc sec61α

Extracted rough microsomes (RM) from snap-frozen pancreatic tissue contain ER translocon subunits and associating factors. Western blot analysis of <t>Sec61</t> translocon subunits and ER translocation-associating factors in the extracted RM samples from 17 different dogs. Selected proteins were identified by western blotting with conditions optimized for each individual primary antibody. Representative images are shown for the following proteins: α subunit of the Sec61 translocon (predicted MW 52 kDa), β subunit of the Sec61 translocon (MW 10 kDa), α subunit of TRAP (MW 32 kDa), BiP (MW 72 kDa), and 60S ribosomal protein L26 (RPL26; MW 17 kDa). The numbers at the top of the figure refer to the ID of each dog sample as in <xref ref-type=

Table 3 . The first and last lanes show the MW marker (in kDa). " width="250" height="auto" />
Sec61α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Rough microsomes isolated from snap-frozen canine pancreatic tissue retain their co-translational translocation functionality"

Article Title: Rough microsomes isolated from snap-frozen canine pancreatic tissue retain their co-translational translocation functionality

Journal: Biology Methods & Protocols

doi: 10.1093/biomethods/bpaf044

Table 3 . The first and last lanes show the MW marker (in kDa). " title="... subunits and associating factors. Western blot analysis of Sec61 translocon subunits and ER translocation-associating factors in the ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: Extracted rough microsomes (RM) from snap-frozen pancreatic tissue contain ER translocon subunits and associating factors. Western blot analysis of Sec61 translocon subunits and ER translocation-associating factors in the extracted RM samples from 17 different dogs. Selected proteins were identified by western blotting with conditions optimized for each individual primary antibody. Representative images are shown for the following proteins: α subunit of the Sec61 translocon (predicted MW 52 kDa), β subunit of the Sec61 translocon (MW 10 kDa), α subunit of TRAP (MW 32 kDa), BiP (MW 72 kDa), and 60S ribosomal protein L26 (RPL26; MW 17 kDa). The numbers at the top of the figure refer to the ID of each dog sample as in Table 3 . The first and last lanes show the MW marker (in kDa).

Techniques Used: Western Blot, Translocation Assay, Marker

Image Search Results

Ca 2 + ‐mediated dialogue between lysosome and ER at MCSs. A) Flow cytometric analysis of Annexin V‐FITC/PI‐PerCP‐stained cell lines of Kasumi‐1, U937, MV4‐11, SKNO‐1, ME‐1, and THP1 treated with 15 µ m of LW‐213 for 1, 3, 6, 9, and 12 h., * p < 0.05, ** p < 0.01, *** p < 0.001 compared to 0 h group. B) The THP1, SKNO‐1 and MV4‐11 cells were exposed to 15 µM of LW‐213 for 0.5, 1, 2, 3, 6, 9, and 12 h, respectively. The Ca 2+ indicator Fluo3‐AM measured cytoplasmic Ca 2+ levels in each group of cells, * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to 0 h group. C) The THP1 cells were exposed to 15 µM of LW‐213 with complete medium or Ca 2+ ‐free medium for 1, 2, 3, 6, and 9 h, respectively. The Ca 2+ indicator Fluo3‐AM measured cytoplasmic Ca 2+ levels in each group of cells, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to Complete Medium group. D) Flow cytometric analysis of Annexin V‐FITC/PI‐PerCP‐stained cell line of THP1 treated with 15 µ m of LW‐213 with complete medium or Ca 2+ ‐free medium for 1, 3, 6, and 9 h, ns indicates non‐significant compared to Complete Medium group. E) The THP1 cells were pretreated with 2‐APB (100 µM) for 2 h, then exposed to 15 µ m LW‐213 for 1, 2, 3, 6, and 9 h. The Ca 2+ indicator Fluo3‐AM measured cytoplasmic Ca 2+ levels in each group of cells, * p < 0.05 compared to LW‐213 group. F) Flow cytometric analysis of Annexin V‐FITC/PI‐PerCP‐stained cell line of THP1 treated with 15 µ m of LW‐213 with/without 2‐APB (100 µ m ) for 1, 2, 3, 6, and 9 h, * p < 0.05 compared to LW‐213 group. G) The Vector and shTPCN1 THP1 cells were exposed to 15 µ m LW‐213 for 1, 2, 3, 6, and 9 h, respectively. The Ca 2+ indicator Fluo3‐AM measured cytoplasmic Ca 2+ levels in each group of cells, * p < 0.05, ** p < 0.01 compared to Vector group. H) The Vector and shTPCN1 THP1 cells were exposed to 15 µ m LW‐213 for 6 h, respectively. The Ca 2+ indicator Fluo3‐AM measured cytoplasmic Ca 2+ levels in each group of cells, ** p < 0.01 compared to Vector 15 µ m LW‐213 group. I) Flow cytometric analysis of Annexin V‐FITC/PI‐PerCP‐stained cell lines of Vector or shTPCN1 THP1 treated with 15 µ m of LW‐213 for 1, 2, 3, 6, and 9 h, ** p < 0.01 compared to Vector 15 µ m LW‐213 group. J) The Vector and shTPCN1 THP1 cells were exposed to 15 µ m LW‐213 with/without 2‐APB (100 µ m ) for 1, 2, 3, 6, and 9 h, respectively. The Ca 2+ indicator Fluo3‐AM measured cytoplasmic Ca 2+ levels in each group of cells, * p < 0.05, ** p < 0.01 compared to 15 µ m LW‐213 group. K) Flow cytometric analysis of Annexin V‐FITC/PI‐PerCP‐stained cell lines of Vector or TPCN1‐OE (#1, #2) HeLa treated with 15 µ m of LW‐213 for 3, 6, 9, and 12 h, ** p < 0.01, *** p < 0.001 compared to Vector group.(L) The Vector and TPCN1‐OE (#1, #2) HeLa treated with 15 µM of LW‐213 for 3, 6, 9, and 12 h, respectively. The Ca 2+ indicator Fluo3‐AM measured cytoplasmic Ca 2+ levels in each group of cells, * p < 0.05, ** p < 0.01 compared to Vector group. M) The THP1 cells were treated with 15 µ m of LW‐213 for 6 h. Cells were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue), LAMP1 protein (red), and SEC61α (green). They were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100). N,O) THP1 and MV4‐11 cells transfected with RA‐SEC61β were treated with 15 µ m of LW‐213 for 6 h. Cells were collected and crawled for immunofluorescence staining of LAMP1 protein (green). They were detected by confocal microscopy (Leica Ultra‐High‐Resolution Laser Confocal) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100). Data are shown as Mean ± S.E.M. from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ns indicates non‐significant.

Journal: Advanced Science

Article Title: Activation of Lysosomal Retrograde Transport Triggers TPC1‐IP3R1 Ca 2+ Crosstalk at Lysosome‐ER MCSs Leading to Lethal Depleting of ER Calcium

doi: 10.1002/advs.202415313

Figure Lengend Snippet: Ca 2 + ‐mediated dialogue between lysosome and ER at MCSs. A) Flow cytometric analysis of Annexin V‐FITC/PI‐PerCP‐stained cell lines of Kasumi‐1, U937, MV4‐11, SKNO‐1, ME‐1, and THP1 treated with 15 µ m of LW‐213 for 1, 3, 6, 9, and 12 h., * p < 0.05, ** p < 0.01, *** p < 0.001 compared to 0 h group. B) The THP1, SKNO‐1 and MV4‐11 cells were exposed to 15 µM of LW‐213 for 0.5, 1, 2, 3, 6, 9, and 12 h, respectively. The Ca 2+ indicator Fluo3‐AM measured cytoplasmic Ca 2+ levels in each group of cells, * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to 0 h group. C) The THP1 cells were exposed to 15 µM of LW‐213 with complete medium or Ca 2+ ‐free medium for 1, 2, 3, 6, and 9 h, respectively. The Ca 2+ indicator Fluo3‐AM measured cytoplasmic Ca 2+ levels in each group of cells, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to Complete Medium group. D) Flow cytometric analysis of Annexin V‐FITC/PI‐PerCP‐stained cell line of THP1 treated with 15 µ m of LW‐213 with complete medium or Ca 2+ ‐free medium for 1, 3, 6, and 9 h, ns indicates non‐significant compared to Complete Medium group. E) The THP1 cells were pretreated with 2‐APB (100 µM) for 2 h, then exposed to 15 µ m LW‐213 for 1, 2, 3, 6, and 9 h. The Ca 2+ indicator Fluo3‐AM measured cytoplasmic Ca 2+ levels in each group of cells, * p < 0.05 compared to LW‐213 group. F) Flow cytometric analysis of Annexin V‐FITC/PI‐PerCP‐stained cell line of THP1 treated with 15 µ m of LW‐213 with/without 2‐APB (100 µ m ) for 1, 2, 3, 6, and 9 h, * p < 0.05 compared to LW‐213 group. G) The Vector and shTPCN1 THP1 cells were exposed to 15 µ m LW‐213 for 1, 2, 3, 6, and 9 h, respectively. The Ca 2+ indicator Fluo3‐AM measured cytoplasmic Ca 2+ levels in each group of cells, * p < 0.05, ** p < 0.01 compared to Vector group. H) The Vector and shTPCN1 THP1 cells were exposed to 15 µ m LW‐213 for 6 h, respectively. The Ca 2+ indicator Fluo3‐AM measured cytoplasmic Ca 2+ levels in each group of cells, ** p < 0.01 compared to Vector 15 µ m LW‐213 group. I) Flow cytometric analysis of Annexin V‐FITC/PI‐PerCP‐stained cell lines of Vector or shTPCN1 THP1 treated with 15 µ m of LW‐213 for 1, 2, 3, 6, and 9 h, ** p < 0.01 compared to Vector 15 µ m LW‐213 group. J) The Vector and shTPCN1 THP1 cells were exposed to 15 µ m LW‐213 with/without 2‐APB (100 µ m ) for 1, 2, 3, 6, and 9 h, respectively. The Ca 2+ indicator Fluo3‐AM measured cytoplasmic Ca 2+ levels in each group of cells, * p < 0.05, ** p < 0.01 compared to 15 µ m LW‐213 group. K) Flow cytometric analysis of Annexin V‐FITC/PI‐PerCP‐stained cell lines of Vector or TPCN1‐OE (#1, #2) HeLa treated with 15 µ m of LW‐213 for 3, 6, 9, and 12 h, ** p < 0.01, *** p < 0.001 compared to Vector group.(L) The Vector and TPCN1‐OE (#1, #2) HeLa treated with 15 µM of LW‐213 for 3, 6, 9, and 12 h, respectively. The Ca 2+ indicator Fluo3‐AM measured cytoplasmic Ca 2+ levels in each group of cells, * p < 0.05, ** p < 0.01 compared to Vector group. M) The THP1 cells were treated with 15 µ m of LW‐213 for 6 h. Cells were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue), LAMP1 protein (red), and SEC61α (green). They were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100). N,O) THP1 and MV4‐11 cells transfected with RA‐SEC61β were treated with 15 µ m of LW‐213 for 6 h. Cells were collected and crawled for immunofluorescence staining of LAMP1 protein (green). They were detected by confocal microscopy (Leica Ultra‐High‐Resolution Laser Confocal) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100). Data are shown as Mean ± S.E.M. from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ns indicates non‐significant.

Article Snippet: Primary antibodies for TPC1 (23758‐1‐AP, RRID:AB_2879317), RAB7 (55469‐1‐AP, RRID:AB_11182831), ARL8B (13049‐1‐AP, RRID: AB_2^059000), TBC1D15 (17252‐1‐AP, RRID:AB_2878370), LC3 (14600‐1‐AP, RRID: AB_2137737), LIMP2 (27102‐1‐AP, RRID: AB_2880756), LAMP1 (67300‐1‐Ig, RRID:AB_2882564), SEC61α (24935‐1‐AP, RRID: AB_2879807), STIM1 (11565‐1‐AP, RRID: AB_2302808), ORAI1 (66223‐1‐Ig, RRID: AB_2881614), mTOR (66888‐1‐Ig, RRID: AB_2882219), CTSB (12216‐1‐AP, RRID: AB_2086929), SERCA2 (67248‐1‐Ig, RRID: AB_2882525), SERCA3 (13619‐1‐AP, RRID: AB_2061448), GRP78 (11587‐1‐AP, RRID: AB_2119855), LAMP2 (66301‐1‐Ig, RRID: AB_2881684), β‐Actin (66009‐1‐Ig, RRID: AB_2687938), GAPDH ( 60004‐1‐Ig, RRID: AB_2107436), CTSD (21327‐1‐AP, RRID: AB_10733646), CHOP (15204‐1‐AP, RRID: AB_2292610) were obtained from Proteintech Technology (Wuhan, China).

Techniques: Staining, Plasmid Preparation, Immunofluorescence, Confocal Microscopy, Software, Transfection

Augmented retrograde transport of lysosomes enhances Ca 2+ crosstalk. A,B) The THP1 cells were exposed to LW‐213 (15 µ m ) for 6 h. The Total RNA was extracted from the cells for RNA‐seq analysis. C,D) The THP1 cells were exposed to LW‐213 (15 µ m ) for 6 h. The distribution of lysosomes was examined by bio‐transmission electron microscopy, as indicated by the yellow arrow in the Figure, *** p < 0.001 compared to 0 µ m LW‐213 group. E,F) The HCT‐116 and HeLa cells were treated with 15 µ m of LW‐213 for 6 h. Cells were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue) and LAMP1 protein (red). They were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100), * p < 0.05, ** p < 0.01 compared to 0 µ m LW‐213 group. G–I) The THP1 and HeLa cells were treated with 15 µ m of LW‐213 for 6 h. Cells were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue) and LAMP1 protein (green). They were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100), ns compared to 15 µ m LW‐213 group. J) Flow cytometric analysis of Annexin V‐FITC/PI‐PerCP‐stained cell line of THP1 treated with 15 µ m of LW‐213 with/without MβCD (0.05, 0.1, and 5 m m ) for 9 h, ns compared to 15 µM LW‐213 group. K,L) The HeLa and shRAB7A or OE‐TBC1D15 HeLa cells were treated with 15 µ m of LW‐213 for 6 h. Cells were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue) and LAMP1 protein (red). They were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100). * p < 0.05, ** p < 0.01, ns compared to 0 µ m LW‐213 group, # p < 0.05, ## p < 0.01 compared to Vector 0 µ m LW‐213 group. (M) The THP1 cells were exposed to LW‐213 (15 µ m ) and CHX (50 µ m ) for 3, 6, and 9 h. (N) The THP1 cells were exposed to LW‐213 (15 µ m ) and BAF‐A1 (50 n m ) or MG‐132(15 µ m ) for 9 h. O) The THP1 and MV4‐11 cells were treated with 15 µ m of LW‐213 with BAF‐A1 (50 n m ) for 6 h. Cells were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue), LAMP1 protein (red) and SEC61α (green). THP1 cells were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100). MV4‐11 cells were detected by confocal microscopy (Leica Ultra‐High‐Resolution Laser Confocal) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type). ** p < 0.01, ns compared to 15 µ m LW‐213 group. P) Flow cytometric analysis of Mag‐Fluo4‐AM‐stained cell line of THP1 treated with 15 µ m of LW‐213 with/without BAF‐A1 (50 n m ) for 1, 2, 3, 6, and 9 h. * p < 0.05, ** p < 0.01 compared to 0 h LW‐213 group, # p < 0.05 compared to 9 h LW‐213 group. Q,R) Flow cytometric analysis of Annexin V‐FITC/PI‐PerCP‐stained cell line of Kasumi‐1, THP1 and MV4‐11 treated with 15 µ m of LW‐213 with/without BAF‐A1 (50 n m ) for 9 h. *** p < 0.001 compared to 15 µ m LW‐213 group. Data are shown as Mean ± S.E.M. from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ns indicates non‐significant.

Journal: Advanced Science

Article Title: Activation of Lysosomal Retrograde Transport Triggers TPC1‐IP3R1 Ca 2+ Crosstalk at Lysosome‐ER MCSs Leading to Lethal Depleting of ER Calcium

doi: 10.1002/advs.202415313

Figure Lengend Snippet: Augmented retrograde transport of lysosomes enhances Ca 2+ crosstalk. A,B) The THP1 cells were exposed to LW‐213 (15 µ m ) for 6 h. The Total RNA was extracted from the cells for RNA‐seq analysis. C,D) The THP1 cells were exposed to LW‐213 (15 µ m ) for 6 h. The distribution of lysosomes was examined by bio‐transmission electron microscopy, as indicated by the yellow arrow in the Figure, *** p < 0.001 compared to 0 µ m LW‐213 group. E,F) The HCT‐116 and HeLa cells were treated with 15 µ m of LW‐213 for 6 h. Cells were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue) and LAMP1 protein (red). They were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100), * p < 0.05, ** p < 0.01 compared to 0 µ m LW‐213 group. G–I) The THP1 and HeLa cells were treated with 15 µ m of LW‐213 for 6 h. Cells were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue) and LAMP1 protein (green). They were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100), ns compared to 15 µ m LW‐213 group. J) Flow cytometric analysis of Annexin V‐FITC/PI‐PerCP‐stained cell line of THP1 treated with 15 µ m of LW‐213 with/without MβCD (0.05, 0.1, and 5 m m ) for 9 h, ns compared to 15 µM LW‐213 group. K,L) The HeLa and shRAB7A or OE‐TBC1D15 HeLa cells were treated with 15 µ m of LW‐213 for 6 h. Cells were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue) and LAMP1 protein (red). They were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100). * p < 0.05, ** p < 0.01, ns compared to 0 µ m LW‐213 group, # p < 0.05, ## p < 0.01 compared to Vector 0 µ m LW‐213 group. (M) The THP1 cells were exposed to LW‐213 (15 µ m ) and CHX (50 µ m ) for 3, 6, and 9 h. (N) The THP1 cells were exposed to LW‐213 (15 µ m ) and BAF‐A1 (50 n m ) or MG‐132(15 µ m ) for 9 h. O) The THP1 and MV4‐11 cells were treated with 15 µ m of LW‐213 with BAF‐A1 (50 n m ) for 6 h. Cells were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue), LAMP1 protein (red) and SEC61α (green). THP1 cells were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100). MV4‐11 cells were detected by confocal microscopy (Leica Ultra‐High‐Resolution Laser Confocal) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type). ** p < 0.01, ns compared to 15 µ m LW‐213 group. P) Flow cytometric analysis of Mag‐Fluo4‐AM‐stained cell line of THP1 treated with 15 µ m of LW‐213 with/without BAF‐A1 (50 n m ) for 1, 2, 3, 6, and 9 h. * p < 0.05, ** p < 0.01 compared to 0 h LW‐213 group, # p < 0.05 compared to 9 h LW‐213 group. Q,R) Flow cytometric analysis of Annexin V‐FITC/PI‐PerCP‐stained cell line of Kasumi‐1, THP1 and MV4‐11 treated with 15 µ m of LW‐213 with/without BAF‐A1 (50 n m ) for 9 h. *** p < 0.001 compared to 15 µ m LW‐213 group. Data are shown as Mean ± S.E.M. from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ns indicates non‐significant.

Techniques: RNA Sequencing, Transmission Assay, Electron Microscopy, Immunofluorescence, Staining, Confocal Microscopy, Software, Plasmid Preparation

Validation of the anti‐tumor efficacy of LW‐213 by targeting LIMP2 in vivo. A,B) Tumor volume was measured every day and quantified as 0.5 × length × width × width. The volume was expressed as mean ± SD (n = 7) and represented as tumor volume–time curves to show. *** p < 0.001 compared to Control group, ### p < 0.001 compared to SCARB2 CTRL LW‐213 (15 mg kg −1 ) group. C) After 10 days of administration, mice were sacrificed, and tumors were weighted. The representative tumors were shown. Tumor weight in each group was expressed as mean ± SD (n = 7). ** p < 0.01, *** p < 0.001 compared to SCARB2 CTRL LW‐213 (0 mg kg −1 ) group, ### p < 0.001 compared to SCARB2 CTRL LW‐213 (15 mg kg −1 ) group. D,E) Tumor tissue sections were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue), Galectin 3 (green) and BrightRed (red). They were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100). F) The expression levels of GRP78 and CHOP were analyzed in tumor tissue protein by western blot. β‐actin was used as a loading control. G) Tumor tissue sections were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue) and CHOP (green). They were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100). H) Tumor tissue sections were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue), SEC61α (red) and LAMP1 (green). They were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100). I) H&E staining for toxicity detection of BALB/c nude mice organs. Data are shown as Mean ± S.E.M. from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ns indicates non‐significant.

Journal: Advanced Science

Article Title: Activation of Lysosomal Retrograde Transport Triggers TPC1‐IP3R1 Ca 2+ Crosstalk at Lysosome‐ER MCSs Leading to Lethal Depleting of ER Calcium

doi: 10.1002/advs.202415313

Figure Lengend Snippet: Validation of the anti‐tumor efficacy of LW‐213 by targeting LIMP2 in vivo. A,B) Tumor volume was measured every day and quantified as 0.5 × length × width × width. The volume was expressed as mean ± SD (n = 7) and represented as tumor volume–time curves to show. *** p < 0.001 compared to Control group, ### p < 0.001 compared to SCARB2 CTRL LW‐213 (15 mg kg −1 ) group. C) After 10 days of administration, mice were sacrificed, and tumors were weighted. The representative tumors were shown. Tumor weight in each group was expressed as mean ± SD (n = 7). ** p < 0.01, *** p < 0.001 compared to SCARB2 CTRL LW‐213 (0 mg kg −1 ) group, ### p < 0.001 compared to SCARB2 CTRL LW‐213 (15 mg kg −1 ) group. D,E) Tumor tissue sections were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue), Galectin 3 (green) and BrightRed (red). They were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100). F) The expression levels of GRP78 and CHOP were analyzed in tumor tissue protein by western blot. β‐actin was used as a loading control. G) Tumor tissue sections were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue) and CHOP (green). They were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100). H) Tumor tissue sections were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue), SEC61α (red) and LAMP1 (green). They were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100). I) H&E staining for toxicity detection of BALB/c nude mice organs. Data are shown as Mean ± S.E.M. from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ns indicates non‐significant.

Techniques: Biomarker Discovery, In Vivo, Control, Immunofluorescence, Staining, Confocal Microscopy, Software, Expressing, Western Blot

Table 3 . The first and last lanes show the MW marker (in kDa). " width="100%" height="100%">

Journal: Biology Methods & Protocols

Article Title: Rough microsomes isolated from snap-frozen canine pancreatic tissue retain their co-translational translocation functionality

doi: 10.1093/biomethods/bpaf044

Figure Lengend Snippet: Extracted rough microsomes (RM) from snap-frozen pancreatic tissue contain ER translocon subunits and associating factors. Western blot analysis of Sec61 translocon subunits and ER translocation-associating factors in the extracted RM samples from 17 different dogs. Selected proteins were identified by western blotting with conditions optimized for each individual primary antibody. Representative images are shown for the following proteins: α subunit of the Sec61 translocon (predicted MW 52 kDa), β subunit of the Sec61 translocon (MW 10 kDa), α subunit of TRAP (MW 32 kDa), BiP (MW 72 kDa), and 60S ribosomal protein L26 (RPL26; MW 17 kDa). The numbers at the top of the figure refer to the ID of each dog sample as in Table 3 . The first and last lanes show the MW marker (in kDa).

Article Snippet: Sec61α , Cell Signaling Technology , Cat #14867, AB_2798635.

Techniques: Western Blot, Translocation Assay, Marker

Overview of the Sec61/TRAP/ribosome complex structure highlighting the interaction sites of TRAP with ribosome (ribosome‐binding site, RBS1, RBS2, and RBS3, indicated with orange arrows) and TRAP with Sec61 complex (translocon‐binding site, TBS1, TBS2, and TBS3, indicated with black arrows). Transmembrane domains of TRAPβ and TRAPδ interact with the transmembrane helices (TM1 and TM3) of TRAPγ to form a trimeric complex. TRAPα traverses the membrane diagonally away from the TRAPβ, TRAPδ, and TRAPγ complex and forms a connection with the backside of Sec61γ on the cytosolic part of Sec61. Identified interprotein crosslink T1 is indicated in green. Formation of the trimeric TRAP complex among TRAPα, TRAPβ, and TRAPδ in the lumenal region of the ER. Data information: TRAP subunits colored as TRAPα:cyan, TRAPβ:pink, TRAPδ:green, and TRAPγ:yellow; Sec61 complex colored as Sec61α:light orange, Sec61β:gray, and Sec61γ:red. 28S rRNA in light green and 5.8S rRNA highlighted in green. All the ribosomal proteins are highlighted in different shades of gray color. Protein/ribosome structures were rendered with ChimeraX, and the schematics were created with BioRender.com .

Journal: EMBO Reports

Article Title: Molecular view of ER membrane remodeling by the Sec61/ TRAP translocon

doi: 10.15252/embr.202357910

Figure Lengend Snippet: Overview of the Sec61/TRAP/ribosome complex structure highlighting the interaction sites of TRAP with ribosome (ribosome‐binding site, RBS1, RBS2, and RBS3, indicated with orange arrows) and TRAP with Sec61 complex (translocon‐binding site, TBS1, TBS2, and TBS3, indicated with black arrows). Transmembrane domains of TRAPβ and TRAPδ interact with the transmembrane helices (TM1 and TM3) of TRAPγ to form a trimeric complex. TRAPα traverses the membrane diagonally away from the TRAPβ, TRAPδ, and TRAPγ complex and forms a connection with the backside of Sec61γ on the cytosolic part of Sec61. Identified interprotein crosslink T1 is indicated in green. Formation of the trimeric TRAP complex among TRAPα, TRAPβ, and TRAPδ in the lumenal region of the ER. Data information: TRAP subunits colored as TRAPα:cyan, TRAPβ:pink, TRAPδ:green, and TRAPγ:yellow; Sec61 complex colored as Sec61α:light orange, Sec61β:gray, and Sec61γ:red. 28S rRNA in light green and 5.8S rRNA highlighted in green. All the ribosomal proteins are highlighted in different shades of gray color. Protein/ribosome structures were rendered with ChimeraX, and the schematics were created with BioRender.com .

Article Snippet: Western blotting analysis of the solubilized ribosome/Sec61/TRAP complex was done with the same SDS–PAGE gels as above and the used primary antibodies were 1:1,000‐diluted anti‐TRAPα antibody Fons et al , 1:1,000‐diluted anti‐Sec61α antibody (NB120‐15575, Novus biologics), and 1:500‐diluted anti‐RPL18A (14653, Proteintech), and the used secondary antibody was 1:10,000‐diluted IRDye® 800CW goat anti‐rabbit IgG secondary antibody (LI‐COR Biosciences).

Techniques: Binding Assay, Membrane

Snapshot of the initial conformation of the simulation system containing the Sec61/TRAP complex together with parts of the ribosome that interact with Sec61 or TRAP subunits. The distal parts of ribosome are restrained to model its large size without the need to model the entire ribosome. TRAP subunits are shown in green (TRAPα), yellow (TRAPβ), blue (TRAPγ), and orange (TRAPδ), whereas Sec61 subunits are shown in pink (Sec61α), cyan (Sec61β), or red (Sec61γ). The ribosomal proteins and RNA fragments included are drawn in gray. The lipids are shown in silver with gray head groups, and cholesterol in white. The extent of the simulation cell is highlighted by the transparent surface. The lipid hydrogens, water molecules, and ions are not rendered for clarity. Root mean square deviation (RMSD) of the TRAP and Sec61 structures when simulated in different assemblies. Sec61 is always stable, yet TRAP conformation shows significant variations in the absence of ribosomal anchoring. Quantitative characterization of membrane perturbations using g_lomepro (Gapsys et al , ). The vertical shift of the lipid phosphorus atoms. The profiles were calculated parallel to the axis connecting Sec61 and TRAP and perpendicular to it. Darker lines show the upper (cytosolic) leaflet and lighter ones the lower (lumenal) leaflet. The extent of the protein TM regions is highlighted. Membrane thickness is calculated as the difference between the phosphorus profiles of the two leaflets in (C). Local membrane ordering calculated as the average of the deuterium order parameters of carbons 2–15 in the palmitate chains of phospholipids. The perturbation of the membrane in the simulation containing Sec61/TRAP anchored by the ribosome contacts. The average positions of the phosphorus atoms are shown by the colored surface cut at the protein location. The color depicts local thickness, ranging from 37 Å (blue) to 43 Å (red). Average of the protein‐free control simulation was 41.8 ± 0.6 Å. Lipid flip–flops as a proxy to membrane perturbation and permeabilization. The cumulative POPC flip–flops in the coarse‐grained simulations. In simulations with individual TRAP subunits, no flip–flops were observed, but they are promoted by the bundle of TRAPβ, TRAPγ, and TRAPδ TM domains. Sec61 alone has a minor effect, but together with TRAP the lensing effect significantly accelerates flip–flops. The distance of the lateral gate helices TM2 and TM7 in the atomistic simulations. The presence of TRAP seems to help maintain the gate in a more open conformation. The section of the cryo‐ET map EMD‐0084 (Pfeffer et al , ; Martinez‐Sanchez et al , ) in the same orientation and positioning as panels (J–L) to highlight Sec61 and TRAP positioning. Leaflet shapes are demonstrated by the height (color) with respect to the center that is set to 0. The cytosolic leaflet is mildly curved, corresponding to the overall microsome shape, and the height shows a change of ∼ 10 Å at a radial distance of ∼ 150 Å. The lumenal leaflet shows a change of ∼ 20 Å over the lateral distance of ∼ 150 Å, indicating a significant and localized curvature. The cytosolic leaflet has localized negative mean curvature, which in the protein vicinity (within 40 Å from the center) corresponds to a radius of curvature of ∼ 320 Å, in line with our MD predictions. The local high curvature is absent in the cytosolic leaflet, and the average radius of curvature of 1,300 Å likely corresponds to a typical microsome size in the sample. The local thickness shows significant membrane thinning from the average value of 34 to ∼ 29 Å in the protein vicinity.

Journal: EMBO Reports

Article Title: Molecular view of ER membrane remodeling by the Sec61/ TRAP translocon

doi: 10.15252/embr.202357910

Figure Lengend Snippet: Snapshot of the initial conformation of the simulation system containing the Sec61/TRAP complex together with parts of the ribosome that interact with Sec61 or TRAP subunits. The distal parts of ribosome are restrained to model its large size without the need to model the entire ribosome. TRAP subunits are shown in green (TRAPα), yellow (TRAPβ), blue (TRAPγ), and orange (TRAPδ), whereas Sec61 subunits are shown in pink (Sec61α), cyan (Sec61β), or red (Sec61γ). The ribosomal proteins and RNA fragments included are drawn in gray. The lipids are shown in silver with gray head groups, and cholesterol in white. The extent of the simulation cell is highlighted by the transparent surface. The lipid hydrogens, water molecules, and ions are not rendered for clarity. Root mean square deviation (RMSD) of the TRAP and Sec61 structures when simulated in different assemblies. Sec61 is always stable, yet TRAP conformation shows significant variations in the absence of ribosomal anchoring. Quantitative characterization of membrane perturbations using g_lomepro (Gapsys et al , ). The vertical shift of the lipid phosphorus atoms. The profiles were calculated parallel to the axis connecting Sec61 and TRAP and perpendicular to it. Darker lines show the upper (cytosolic) leaflet and lighter ones the lower (lumenal) leaflet. The extent of the protein TM regions is highlighted. Membrane thickness is calculated as the difference between the phosphorus profiles of the two leaflets in (C). Local membrane ordering calculated as the average of the deuterium order parameters of carbons 2–15 in the palmitate chains of phospholipids. The perturbation of the membrane in the simulation containing Sec61/TRAP anchored by the ribosome contacts. The average positions of the phosphorus atoms are shown by the colored surface cut at the protein location. The color depicts local thickness, ranging from 37 Å (blue) to 43 Å (red). Average of the protein‐free control simulation was 41.8 ± 0.6 Å. Lipid flip–flops as a proxy to membrane perturbation and permeabilization. The cumulative POPC flip–flops in the coarse‐grained simulations. In simulations with individual TRAP subunits, no flip–flops were observed, but they are promoted by the bundle of TRAPβ, TRAPγ, and TRAPδ TM domains. Sec61 alone has a minor effect, but together with TRAP the lensing effect significantly accelerates flip–flops. The distance of the lateral gate helices TM2 and TM7 in the atomistic simulations. The presence of TRAP seems to help maintain the gate in a more open conformation. The section of the cryo‐ET map EMD‐0084 (Pfeffer et al , ; Martinez‐Sanchez et al , ) in the same orientation and positioning as panels (J–L) to highlight Sec61 and TRAP positioning. Leaflet shapes are demonstrated by the height (color) with respect to the center that is set to 0. The cytosolic leaflet is mildly curved, corresponding to the overall microsome shape, and the height shows a change of ∼ 10 Å at a radial distance of ∼ 150 Å. The lumenal leaflet shows a change of ∼ 20 Å over the lateral distance of ∼ 150 Å, indicating a significant and localized curvature. The cytosolic leaflet has localized negative mean curvature, which in the protein vicinity (within 40 Å from the center) corresponds to a radius of curvature of ∼ 320 Å, in line with our MD predictions. The local high curvature is absent in the cytosolic leaflet, and the average radius of curvature of 1,300 Å likely corresponds to a typical microsome size in the sample. The local thickness shows significant membrane thinning from the average value of 34 to ∼ 29 Å in the protein vicinity.

Techniques: Membrane, Tomography

Cryo‐EM structure of our Sec61/TRAP complex and the structure of OST‐A (PDB ID: 6S7O) complex modeled in the cryo‐ET density of Sec61/TRAP and the OST complex (EMD‐3068), surface (right), and cartoon (left) representation. TRAP subunits colored as TRAPα:cyan, TRAPβ:pink, TRAPδ:green, and TRAPγ: yellow, and Sec61 complex colored as Sec61α:light orange, Sec61β:gray, and Sec61γ:red. Most of the OST subunits are colored in gray except STT3A and RbII which lie in proximity to the lumenal domains of the TRAP complex and are colored green and light green, respectively. The glycosylation active site of the STT3A domain is highlighted with an orange circle. Protein/ribosome structures were rendered with ChimeraX, and the schematics were created with BioRender.com . Targeting of the ribosome–nascent chain complex (ribosome in gray, nascent chain's signal peptide in red, and its mature chain in black) is carried out by SRP and SRP receptor (in blue) (1). Docking of the ribosome induces a conformational change in the TRAP/Sec61 complex (TRAP in turquoise and Sec61 in light brown), resulting in membrane perturbation which increases the fluidity of the local lipid environment about the Sec61 lateral gate (2). After successful lateral gate engagement and plug displacement, nascent polypeptide is exposed to the ER lumen, where transient interactions with the negatively charged TRAP α flexible N‐terminal loop (in black) may encourage correct topology and complete lateral gate intercalation of the signal peptide (3). The TRAP α lumenal domain is proximal to the lumenal exit of Sec61, and it may directly bind the nascent polypeptide, serving to prevent back diffusion of translocation inefficient polypeptide sequences (4). Finally, interaction with TRAP α lumenal domain may also serve to transiently restrict the nascent polypeptide for presentation to downstream processing events.

Journal: EMBO Reports

Article Title: Molecular view of ER membrane remodeling by the Sec61/ TRAP translocon

doi: 10.15252/embr.202357910

Figure Lengend Snippet: Cryo‐EM structure of our Sec61/TRAP complex and the structure of OST‐A (PDB ID: 6S7O) complex modeled in the cryo‐ET density of Sec61/TRAP and the OST complex (EMD‐3068), surface (right), and cartoon (left) representation. TRAP subunits colored as TRAPα:cyan, TRAPβ:pink, TRAPδ:green, and TRAPγ: yellow, and Sec61 complex colored as Sec61α:light orange, Sec61β:gray, and Sec61γ:red. Most of the OST subunits are colored in gray except STT3A and RbII which lie in proximity to the lumenal domains of the TRAP complex and are colored green and light green, respectively. The glycosylation active site of the STT3A domain is highlighted with an orange circle. Protein/ribosome structures were rendered with ChimeraX, and the schematics were created with BioRender.com . Targeting of the ribosome–nascent chain complex (ribosome in gray, nascent chain's signal peptide in red, and its mature chain in black) is carried out by SRP and SRP receptor (in blue) (1). Docking of the ribosome induces a conformational change in the TRAP/Sec61 complex (TRAP in turquoise and Sec61 in light brown), resulting in membrane perturbation which increases the fluidity of the local lipid environment about the Sec61 lateral gate (2). After successful lateral gate engagement and plug displacement, nascent polypeptide is exposed to the ER lumen, where transient interactions with the negatively charged TRAP α flexible N‐terminal loop (in black) may encourage correct topology and complete lateral gate intercalation of the signal peptide (3). The TRAP α lumenal domain is proximal to the lumenal exit of Sec61, and it may directly bind the nascent polypeptide, serving to prevent back diffusion of translocation inefficient polypeptide sequences (4). Finally, interaction with TRAP α lumenal domain may also serve to transiently restrict the nascent polypeptide for presentation to downstream processing events.

Techniques: Cryo-EM Sample Prep, Tomography, Membrane, Diffusion-based Assay, Translocation Assay

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1) Product Images from "Rough microsomes isolated from snap-frozen canine pancreatic tissue retain their co-translational translocation functionality"

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