gal 3 bp 2226 gab r d systems  (R&D Systems)

Journal: Cell Death Discovery

Article Title: RNF8 enhances the sensitivity of PD-L1 inhibitor against melanoma through ubiquitination of galectin-3 in stroma

doi: 10.1038/s41420-023-01500-3

Figure Lengend Snippet: A Gene set enrichment analysis showing changed pathways in RNF8 deficieny cells. B Gene set enrichment analysis showing changed biological process in RNF8 deficieny cells. C Volcano plot of ubiquitination modifications omics in RNF8 deficiency cells. D Western blot of gal-3 in tumor-beared RNF8 +/+ and RNF8 −/− mice. E Western blot of gal-3 in center and periphery of tumor-beared RNF8 +/+ and RNF8 −/− mice. F The technical routine of gal-3 binding with IL-12 or IFN-γ. G IL-12 and ( H ) IFN-γ content in the supernatant were measured by ELISA after incubation with gal-3-coated beads in the presence or absence of 100 mM lactose (left) or 50 mM LAC (right). The human melanoma A375 cells and human lung cancer A549 cells were treated with 0.2 g/L Na 2 SO 3 . I – K Gal-3, HIF-1α, p-IκBα, NF-κB, and p-NF-κB expression in A375 and A549 were detected by western blot at different time after treating with Na 2 SO 3 . L Western blot investigation of HIF-1α in tumor center and periphery in RNF8 +/+ and RNF8 −/− mice. M The schematic diagram of a mechanism related to gal-3 in TME. All values were presented as the mean ± SD ( n = 3). Student’s t -test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Dynabeads M-280 Streptavidin (11-205-D, Invitrogen) were incubated with saturating amounts of recombinant human gal-3 (10289-H08H1, sinobiological) overnight at 4 °C.

Techniques: Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Expressing

Journal: Cell Death Discovery

Article Title: RNF8 enhances the sensitivity of PD-L1 inhibitor against melanoma through ubiquitination of galectin-3 in stroma

doi: 10.1038/s41420-023-01500-3

Figure Lengend Snippet: A Western blot investigation of RNF8 and gal-3 expression in shCtrl and shRNF8 groups in 293 T cell. B RNF8 and gal-3 expression in Ctrl-OV and RNF8-overexpression (RNF8-OV) groups. C A375 cells were transfected with LV-RNF8 for shRNF8 or RNF8-OV. The yellow arrows indicated the successful transfected cells, and the purple arrows indicated the non-transfected cells. D The statistical analysis of gal-3 expression in A375 cells. E Fluorescent staining of endogenous RNF8 and gal-3 distribution in A375 cells. F , G Co-IP analysis of interaction between endogenous RNF8 and gal-3 in 293 T ( F ) and A375 cells ( G ). H , I Co-IP analysis of interaction between exogenous RNF8 and gal-3 in 293 T ( H ) and A375 cells ( I ), wcl whole cell lysate. The values were presented as the mean ± SD ( n = 3). Student’s t -test; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Dynabeads M-280 Streptavidin (11-205-D, Invitrogen) were incubated with saturating amounts of recombinant human gal-3 (10289-H08H1, sinobiological) overnight at 4 °C.

Techniques: Western Blot, Expressing, Over Expression, Transfection, Staining, Co-Immunoprecipitation Assay

Journal: Cell Death Discovery

Article Title: RNF8 enhances the sensitivity of PD-L1 inhibitor against melanoma through ubiquitination of galectin-3 in stroma

doi: 10.1038/s41420-023-01500-3

Figure Lengend Snippet: A CHX (5 μg/mL) was used in 293 T cells to measure gal-3 degradation. B 293 T cells were treated with or without 10 mmol/L MG132 for 4 h. The ubiquitination level of gal-3 was detected. C HA-tagged gal-3 and His-tagged Ub plasmids were co-transfected into 293 T cells for 36 h, followed by cell lysate preparation and IP assay with anti-HA beads followed by immunoblotting with indicated antibodies. D 293 T cells were treated with exogenous RNF8 (shRNF8 or RNF8-OV), gal-3 and Ub, and the ubiquitination of HA was investigated. E – G 293 T cells were transfected with LV-shRNF8 or LV-RNF8-OV, and the ubiquitination ( E ), K48 ( F ), and K63 ( G ) of gal-3 were investigated. H 293 T cells were transfected with gal-3K176R (mutation of gal-3 at lysine 176 site), the expression of gal-3 was investigated. I – K 293 T cells were transfected with Flag-Ctrl, Flag-LGALS3, and Flag-K176R, and the total ubiquitination ( I ), K48 linked ( J ), and K63-linked ( K ) ubiquitination was detected.

Article Snippet: Dynabeads M-280 Streptavidin (11-205-D, Invitrogen) were incubated with saturating amounts of recombinant human gal-3 (10289-H08H1, sinobiological) overnight at 4 °C.

Techniques: Transfection, Western Blot, Mutagenesis, Expressing

Journal: Cell Death Discovery

Article Title: RNF8 enhances the sensitivity of PD-L1 inhibitor against melanoma through ubiquitination of galectin-3 in stroma

doi: 10.1038/s41420-023-01500-3

Figure Lengend Snippet: A , B LAC was injected intratumorally at a single dose of 0.1 μmol per mice after tumor formation, the ratio of tumor weight ( A ), tumor volumes ( B ) were detected. C , D HE staining of implanted melanomas in RNF8 +/+ and RNF8 −/− mice treated with DMSO or LAC. The arrow points to TILs. E , F SPADE analysis of TILs in RNF8 +/+ + DMSO, RNF8 +/+ + LAC, RNF8 −/− + DMSO, and RNF8 −/− + LAC mice. G Analysis of IL-12 by immunohistochemical staining (Supplementary Fig. ) of tumors in above groups. H Analysis of IFN-γ by immunohistochemical staining (Supplementary Fig. ) of tumors in above groups. I Western blot of CD3, gal-3, IL-12, IFN-γ, CXCL-9, and CXCL-10 expression in tumor center and periphery in above groups. The values were presented as the mean ± SD ( n = 3). Student’s t -test; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Dynabeads M-280 Streptavidin (11-205-D, Invitrogen) were incubated with saturating amounts of recombinant human gal-3 (10289-H08H1, sinobiological) overnight at 4 °C.

Techniques: Injection, Staining, Immunohistochemical staining, Western Blot, Expressing