anti ha c29f4 rabbit mab  (Cell Signaling Technology Inc)

Journal: Communications Biology

Article Title: Deacetylation of nuclear AIF provides a braking mechanism for caspase-independent chromatinolysis and necrotic brain injury

doi: 10.1038/s42003-025-08255-w

Figure Lengend Snippet: A SK-N-SH cells treated with MNNG (12 h) or OGD (12 h) were stained for AIF (green) and SIRT1 (red) and viewed using confocal fluorescence microscopy. Nuclei were stained with DAPI (blue). Scale bars indicate 10 µm. Magnification scale bars indicate 1 µm. B , C Binding of AIF and SIRT1 under MNNG or OGD treatment. For MNNG treatment, cells were incubated with a culture medium containing 500 µM MNNG for 20 min, then washed three times with PBS and re-cultured for an additional 12 h in fresh medium without MNNG. For OGD treatment, cells were placed in an I-CONIC hypoxic chamber (95% N 2 ; 5% CO 2 ) with a culture medium lacking glucose for 12 h. The post-nuclear (PN) and nuclear fractions (N) of SK-N-SH cells after MNNG or OGD treatment were prepared, followed by immunoprecipitation of SIRT1 and immunodetection of SIRT1 and AIF as indicated. α-tubulin (the post-nuclear marker) and histone H3 (the nuclear marker) were used to control the quality of fractionation. D SIRT1 physically binds to AIF. Bacterially purified GST or GST-SIRT1 was incubated with His-AIF isolated from E. coli , and the GST pull-down assays were performed, followed by anti-GST and anti-His western blotting analysis. E SIRT1 deacetylase catalytic domain interacts with AIF. The schematic diagrams of SIRT1 full-length (FL) and its deletion mutants are shown in the upper panel. The indicated SIRT1 full-length plasmid and its deletion mutant plasmids were co-transfected with AIF-HA in 293 T cells, followed by anti-Flag immunoprecipitation and subsequent anti-HA western blotting analysis (lower panel). F The NADH and FAD domain of AIF binds to SIRT1. The schematic diagrams of AIF full-length and its deletion mutants are shown in the upper panel. The indicated vector and AIF plasmids were co-transfected with HA-SIRT1 in 293 T cells, and anti-Flag immunoprecipitation was performed followed by anti-HA western blotting (lower panel). The input of HA-SIRT1 is shown in the bottom panel.

Article Snippet: Anti-Acetylated-lysine (Ac-K 2 -100) MultiMabTM Rabbit mAb, acetylated-lysine Mouse mAb (Ac-K-103), and anti-HA-tag (C29F4) rabbit mAb were obtained from Cell Signaling Technology. anti-AIF (A2568), anti-SIRT1 (A19667) rabbit mAb, anti-Flag rabbit mAb (AE092), anti-PARP-1 (A0942) rabbit mAb, and anti-MIF (A1391) rabbit mAb were obtained from ABclonal.

Techniques: Staining, Fluorescence, Microscopy, Binding Assay, Incubation, Cell Culture, Immunoprecipitation, Immunodetection, Marker, Control, Fractionation, Purification, Isolation, Western Blot, Histone Deacetylase Assay, Plasmid Preparation, Mutagenesis, Transfection

Journal: Communications Biology

Article Title: Deacetylation of nuclear AIF provides a braking mechanism for caspase-independent chromatinolysis and necrotic brain injury

doi: 10.1038/s42003-025-08255-w

Figure Lengend Snippet: A SK-N-SH cells were harvested 12 h after MNNG or OGD treatment. For MNNG treatment, cells were incubated with a culture medium containing 500 µM MNNG for 20 min, then re-cultured for another 12 h in fresh medium. The whole-cell lysates were then prepared and immunoprecipitated with anti-AIF antibody and blotted with anti-acetylated lysine antibody as indicated. The acetylation of AIF was analyzed by western blot. B SK-N-SH cells were harvested 12 h after MNNG treatment, then the whole-cell lysates were prepared and immunoprecipitated with anti-acetylation antibody (Ac-K IP) as indicated. The acetylation status of AIF following MNNG treatment was analyzed by western blot using anti-AIF antibody. C 293 T cells were transiently transfected with AIF-Flag, HA-CBP, or Myc-p300 plasmids for 24 h, cells were harvested 12 h after MNNG treatment, then cell lysates were immunoprecipitated with FlAG-M2 beads, followed by western blotting with anti-acetylated lysine, anti-HA, and anti-Myc antibodies. D - E SK-N-SH cells were harvested at the indicated times after MNNG or OGD treatment. The post-nuclear and nuclear extracts from the cells were immunoprecipitated with an anti-AIF antibody and analyzed by western blotting with anti-acetylated lysine and anti-AIF antibodies, respectively. Histone H3 (nuclear marker) and α-tubulin (post-nuclear marker) were used to control for fraction quality and protein loading. F Scramble siRNA (Mock-si) or siRNA against human SIRT1 (SIRT1-si) was transfected into SK-N-SH cells. About 48 h after transfection, cells were treated with MNNG or OGD for another 12 h. Then, cells were harvest and nuclear extracts were immunoprecipitated with an anti-AIF antibody, and acetylated AIF in the nucleus was detected (upper panel). SIRT1 expression was examined by western blotting (lower panel). G 293 T cells were transfected with either a vector or AIF-Flag plasmids were pre-treated with DMSO, SRT1720 (10 µM) or Sirtinol (50 µM) for 12 h followed by 20 min of treatment with 500 µM MNNG. The cells were then maintained in fresh medium for another 12 h. Cell lysates were immunoprecipitated with Flag-M2 beads, followed by western blotting with an anti-acetylated lysine antibody. H 293 T cells co-transfected with indicated plasmids were treated with 500 µM MNNG for 20 min, then the cells were maintained in fresh medium for another 12 h. Cells were lysed and immunoprecipitated with FLAG-M2 beads, followed by western blot analysis. AIF acetylation (ac-AIF) was detected using anti-acetylated lysine or anti-Flag antibodies. I In vitro deacetylation of AIF by SIRT1. Purified AIF from 293 T cells was incubated with recombinant E. coli -expressed GST-SIRT1 wild-type (WT) or GST-SIRT1-H363A protein in the absence or presence of nicotinamide adenine dinucleotide (NAD) (50 mM) or NAM (200 mM) for 2 h at 30 °C. The ac-AIF level was detected using an anti-acetylated lysine antibody.

Article Snippet: Anti-Acetylated-lysine (Ac-K 2 -100) MultiMabTM Rabbit mAb, acetylated-lysine Mouse mAb (Ac-K-103), and anti-HA-tag (C29F4) rabbit mAb were obtained from Cell Signaling Technology. anti-AIF (A2568), anti-SIRT1 (A19667) rabbit mAb, anti-Flag rabbit mAb (AE092), anti-PARP-1 (A0942) rabbit mAb, and anti-MIF (A1391) rabbit mAb were obtained from ABclonal.

Techniques: Incubation, Cell Culture, Immunoprecipitation, Western Blot, Transfection, Marker, Control, Expressing, Plasmid Preparation, In Vitro, Purification, Recombinant