sec61b  (Proteintech)

Journal: bioRxiv

Article Title: Derlin-mediated ERAD of lipid regulator ORMDL3 safeguards mitochondrial function

doi: 10.64898/2026.02.27.708653

Figure Lengend Snippet: (A) Post-nuclear supernatant (PNS), mitochondria, endoplasmic reticulum (ER), and MERCs fractions were isolated from WT, Derlin-2 KO, and Derlin-3 KO cells expressing V5-tagged ORMDL1, ORMDL2, or ORMDL3. Immunoblot analysis was performed using anti-V5 antibodies to detect ORMDL proteins across subcellular fractions. Endogenous Derlin-1 and Derlin-2 were probed, and fraction purity and enrichment were validated using Sec61β as an ER marker, TOMM20 as a mitochondrial marker, and Sigma-1 receptor (Sigma1R) as a MERC marker. (B) MERC enrichment of V5-tagged ORMDL1, ORMDL2, and ORMDL3 was quantified from subcellular fractionation experiments in (A) ORMDL abundance in the MERC fraction was normalized to the ER marker Sec61B (ORMDL/Sec61) to account for ER content within contact sites. Data are shown as mean ± SEM from independent experiments, with individual data points representing biological replicates. Statistical significance was determined by one-way ANOVA with post-hoc multiple-comparison testing. *P < 0.05; ***P < 0.001. (C-D) MERC fractionation and quantification were performed as in (A) and (B), except that ORMDL distribution was analyzed in Derlin-2 KO cells with empty vector and Derlin-2 KO with ectopic expression of Derlin-2-Myc. (E) Images of in situ PLA (indicated in red) monitoring VDAC1-ORMDL3-V5 interaction in WT, Derlin-2 KO, and Derlin-2 KO with ectopic Derlin-2 cells. Scale bar, 10 uM. (F) Quantitative analysis of VDAC1-ORMDL3-V5 signals in WT, Derlin-2 KO, and Derlin-2 KO with ectopic Derlin-2 cells. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple-comparison test. *P < 0.05, **P < 0.01, ****P < 0.0001; ns, not significant.

Article Snippet: F-10 sc-17764), Sec61B (ER; Proteintech 51020-2-AP), and SIGMA1R (MERCS; Santa Cruz Biotech.

Techniques: Isolation, Expressing, Western Blot, Marker, Fractionation, Comparison, Plasmid Preparation, In Situ

Journal: EMBO Reports

Article Title: Sec61β maintains cytoplasmic proteostasis via ARIH1-mediated translational repression upon ER stress

doi: 10.1038/s44319-026-00690-y

Figure Lengend Snippet: ( A ) Gene Ontology (GO) analysis of Derlins-interacting proteins by thapsigargin (Tg) treatment. The bar graph shows the top 10 GO molecular function terms with a false discovery rate of <0.05 calculated from the DAVID online tool. P values were calculated using the modified Fisher’s exact test implemented in DAVID; 37 proteins identified as RNA binding in terms of molecular function are listed in Dataset . ( B ) Interactions of Derlins with Sec61β. HEK293 cells transfected with indicated plasmids and treated with or without 50 nM Tg and 200 nM MG132 for 16 h were immunoprecipitated (IPed) with an anti-Flag antibody and immunoblotted with indicated antibodies. ( C ) Endogenous interaction of Derlin-1 with Sec61β. Immunoprecipitation (IP) with anti-Sec61β antibody or control (Ctrl) IgG using Protein G Sepharose and immunoblotting (IB) with indicated antibodies in HepG2 cells treated with or without 200 nM Tg and/or 500 nM MG132 for 16 h. ( D – F ) IB of ERpQC substrates in HEK293 cells transfected with indicated siRNAs and plasmids and treated with or without 50 nM Tg and 200 nM MG132 for 16 h. All samples were immunoblotted with indicated antibodies. Black arrowhead, signal peptide-uncleaved NHK QQQ ( S NHK QQQ ); white arrowhead, signal peptide-cleaved NHK QQQ ( C NHK QQQ ). ( G ) IB of ERpQC substrate in wild-type (WT) or Derlin-1, -2 , and -3 triple knockout (TKO) HEK293 cells transfected with indicated siRNAs and plasmid for NHK QQQ and treated with or without 50 nM Tg and 200 nM MG132 for 16 h. All samples were immunoblotted with indicated antibodies. Expression levels of S NHK QQQ were calculated and shown as the percentage of S NHK QQQ out of the total amount of NHK QQQ ( S NHK QQQ and C NHK QQQ ). Black arrowhead, S NHK QQQ ; white arrowhead, C NHK QQQ . .

Article Snippet: Rabbit polyclonal anti-Sec61β (IB: 1:500) , Proteintech , Cat. #15087-1-AP; RRID: AB_2186411.

Techniques: Modification, RNA Binding Assay, Transfection, Immunoprecipitation, Control, Western Blot, Triple Knockout, Plasmid Preparation, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Transferrin receptor 1 nuclear translocation facilitates tumor progression via p53-mediated chromatin interactions and genome-wide alterations

doi: 10.1038/s41392-025-02297-6

Figure Lengend Snippet: Identification of the nuclear localization of TfR1 and characterization of its nuclear translocation mechanism. a Representative IHC images (×10 magnification) of TfR1 staining in tumor tissues from different types of cancer ( n = 204). Scale bar = 50 μm. b , c Fractionation of the nuclear and cytoplasmic components of HCT-116 cells in ( b ) and HCT-116 cells transfected with empty vector (EV) or Flag-TfR1 in ( c ). Lamin B1 was used as a nuclear (Nu) loading control. Tubulin was used as non-nuclear fraction (NNF), and whole cell lysate (WCL) loading control. d , e HCT-116 cells were treated with different concentrations of CPZ for 30 min in ( d ) or BFA for 6 h in ( e ) for immunoblot analysis of nuclear and cytoplasmic fractions. The relative abundance of nuclear TfR1 was normalized to that of the CPZ or BFA = 0 controls. Lamin B1 was used as a Nu loading control. Tubulin was used as the loading control for NNF and WCL. f Co-IP analysis of the interactions between Flag-TfR1 and Sec61β. GAPDH was used as a loading control. g HCT-116 cells were transfected with scrambled negative control siRNA (-) or SEC61B -targeted siRNA (+) for 72 h, followed by subcellular fractionation and immunoblot analysis. The relative abundance of nuclear TfR1 was normalized to that of the siRNA (-) control. Lamin B1 and Tubulin were used as equal loading controls for Nu, NNF, and WCL

Article Snippet: These antibodies include anti-TfR1 antibody (Sigma, HPA028598), anti-Lamin B1 antibody (Abcam, ab133741), anti-α-Tubulin antibody (Beyotime, AT819), anti-Calreticulin antibody (Abcam, ab92516), anti-Syntaxin-6 antibody (CST, 2869), anti-importin-β1 antibody (Abcam, ab2811), anti-importin-α antibody (Abcam, ab307438), anti-Transportin-1 antibody (CST, 31452), anti-Sec61β antibody (CST, 14648), anti-GAPDH antibody (ABclonal, AC033), anti-Flag antibody (Sigma, F3165), anti-p53 antibody (Santa Cruz, sc-126), anti-p53 antibody (CST, 2524), anti-p53 antibody (Abcam, ab26), anti-XPC antibody (Santa Cruz, sc-74410), anti-γH2AX antibody (Abcam, ab81299), anti-Ki67 antibody (CST, 9449), mouse IgG (CST, 5415), rabbit IgG (CST, 3900), anti-RS4X antibody (Immunoway, YT4135).

Techniques: Translocation Assay, Staining, Fractionation, Transfection, Plasmid Preparation, Control, Western Blot, Co-Immunoprecipitation Assay, Negative Control