Journal: International Journal of Molecular Sciences

Article Title: Identification of Binding Proteins for TSC22D1 Family Proteins Using Mass Spectrometry

doi: 10.3390/ijms222010913

Figure Lengend Snippet: Identification of TSC-22-binding proteins in cells before and after DNA damage. ( A ): TSC-22-binding protein (arrow) in the nuclear fraction after DNA damage by phleomycin in cells expressing full-length TSC-22. a: Lamin-B1, Heat shock 70-kDa protein 1A/1B, b: Guanine nucleotide-binding protein-like 3 (GNL3), c: Pituitary adenylate cyclase-activating polypeptide type I receptor, d: Charged multivesicular body protein 4a, Histone H1.2, Heterogeneous nuclear ribonucleoprotein A1, rRNA 2′-O-methyltransferase fibrillarin, *Flag-TSC-22. ( B ): TSC22(86)-binding proteins (←) after DNA damage by phleomycin in a whole cell fraction of cells expressing TSC22(86). a: Keratin, type II cytoskeletal 1, Ubiquitin carboxyl-terminal hydrolase 47, Protein tyrosine phosphatase receptor type C-associated protein, b: Keratin, type I cytoskeletal 9, Keratin, type II cytoskeletal 1, c: Histone H1.4, d: Histone H1t, Histone H1.2, e: Actin, cytoplasmic 1, f: Actin, cytoplasmic 1, Histone H1.2, Glyceraldehyde-3-phosphate dehydrogenase, g: Histone H1.2, Dermcidin, h: 40S ribosomal protein S6, Histone H1.2, Dermcidin, 40S ribosomal protein S2, *Flag-TSC22(86).

Article Snippet: After electrophoresis, the blot was prepared by applying an electric current for 7 min using a Bio-Rad Trans-Blot Turbo transfer system and blocked with 2% bovine serum albumin (BSA, Fujifilm, Wako Pure Chemical)-TTBS for 1 h. The blotted membrane was incubated with rabbit anti-human TSC22D1 polyclonal antibody, mouse anti-human histone H1 monoclonal antibody (Genetex, Irvine, CA, USA), and rabbit anti-human GNL3 polyclonal antibody (Genetex) 1000-fold diluted with 2% BSA-TTBS at room temperature for 2 h. Then, washing with TTBS for 10 min was repeated three times, and the blots were incubated with 4000-fold diluted HRP-conjugated anti-rabbit or anti-mouse secondary antibody (Santa Cruz Biotechnology) at room temperature for 1 h, followed by washing with TTBS for 10 min three times and detection using ELC Plus (Thermo Fisher Scientific) and X-ray film (Fuji super RX, Fujifilm, Tokyo, Japan).

Techniques: Binding Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Identification of Binding Proteins for TSC22D1 Family Proteins Using Mass Spectrometry

doi: 10.3390/ijms222010913

Figure Lengend Snippet: Intracellular binding of TSC-22 (TSC22D1-2) with histone H1. IP-Western blotting using nuclear extract after DNA damage in Flag-TSC-22 (TSC22D1-2)-transfected cells. In detection using anti-histone H1 or anti-GNL3 antibody, histone H1 was clearly detected.

Article Snippet: After electrophoresis, the blot was prepared by applying an electric current for 7 min using a Bio-Rad Trans-Blot Turbo transfer system and blocked with 2% bovine serum albumin (BSA, Fujifilm, Wako Pure Chemical)-TTBS for 1 h. The blotted membrane was incubated with rabbit anti-human TSC22D1 polyclonal antibody, mouse anti-human histone H1 monoclonal antibody (Genetex, Irvine, CA, USA), and rabbit anti-human GNL3 polyclonal antibody (Genetex) 1000-fold diluted with 2% BSA-TTBS at room temperature for 2 h. Then, washing with TTBS for 10 min was repeated three times, and the blots were incubated with 4000-fold diluted HRP-conjugated anti-rabbit or anti-mouse secondary antibody (Santa Cruz Biotechnology) at room temperature for 1 h, followed by washing with TTBS for 10 min three times and detection using ELC Plus (Thermo Fisher Scientific) and X-ray film (Fuji super RX, Fujifilm, Tokyo, Japan).

Techniques: Binding Assay, Western Blot, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Identification of Binding Proteins for TSC22D1 Family Proteins Using Mass Spectrometry

doi: 10.3390/ijms222010913

Figure Lengend Snippet: Putative function of TSC-22 (TSC22D1-2) and TSC-22(86) (TSC22D1-3). ( A ): TSC-22 may promote the degradation of histone H1 through this interaction, and replacement of chromatin binding by HMGA2 may cause changes in the expression of genes that regulate cell proliferation and differentiation, apoptosis induction, and cell senescence. ( B ): GNL3 is known as nucleostemin and is found in the nucleolus. GNL3 interacts with p53 and MDM2. TSC-22 (TSC22D1-2) might influence the inhibitory action of GNL3 on tumor cell growth.

Article Snippet: After electrophoresis, the blot was prepared by applying an electric current for 7 min using a Bio-Rad Trans-Blot Turbo transfer system and blocked with 2% bovine serum albumin (BSA, Fujifilm, Wako Pure Chemical)-TTBS for 1 h. The blotted membrane was incubated with rabbit anti-human TSC22D1 polyclonal antibody, mouse anti-human histone H1 monoclonal antibody (Genetex, Irvine, CA, USA), and rabbit anti-human GNL3 polyclonal antibody (Genetex) 1000-fold diluted with 2% BSA-TTBS at room temperature for 2 h. Then, washing with TTBS for 10 min was repeated three times, and the blots were incubated with 4000-fold diluted HRP-conjugated anti-rabbit or anti-mouse secondary antibody (Santa Cruz Biotechnology) at room temperature for 1 h, followed by washing with TTBS for 10 min three times and detection using ELC Plus (Thermo Fisher Scientific) and X-ray film (Fuji super RX, Fujifilm, Tokyo, Japan).

Techniques: Binding Assay, Expressing