%0 Journal Article
%T Stress-induced phosphoprotein 1 acts as a scaffold protein for glycogen synthase kinase-3 beta-mediated phosphorylation of lysine-specific demethylase 1
%A An-Shine Chao
%A Angel Chao
%A Chia-Lung Tsai
%A Chiao-Yun Lin
%A Shih-Ming Jung
%A Tzu-Hao Wang
%J Archive of "Oncogenesis".
%D 2018
%R 10.1038/s41389-018-0040-z
%X a As far as STIP1 is concerned, we used the following Halo-tagged constructs: (1) the full-length (FL) protein, (2) N-terminal deleted halo-tag STIP1 constructs (F3: TPR1 deleted, F2: TPR1-DP1-TPR2A deleted and F1: TPR1-DP1-TPR2A-TPR2B deleted), and (3) C-terminal deleted halo-tag STIP1 constructs (R3: DP2 deleted, R2: TPR2B-DP2 deleted, and R1: DP1-TPR2A-TPR2B-DP2 deleted). The constructs were co-transfected with Flag每LSD1 or HA-GSK3汕 in 293 cells and subsequently purified with Halo-tag resin. The interactions between LSD1 and GSK3汕 were examined with Western blot. b, c 293 cells were co-transfected with (1) FL flag每LSD1, (2) its deleted N-terminal Flag每LSD1 constructs (D1: amino acid 272ˋ852, D2: amino acid 415ˋ852, and D3: amino acid 515ˋ852), (3) its deleted C-terminal Flag每LSD1 constructs (N1: amino acid 1ˋ515, D2: amino acid 1ˋ272), and (4) FL Halo-STIP1 or NTAP每GSK3汕. After purification with a halo-tag resin (Halo-STIP1) or streptavidin beads (NTAP每GSK3汕), co-immunoprecipitated Flag每LSD1 constructs were analyzed with Western blot using an anti-flag tag antibody. c The identification of GSK3汕 domains involved in the interaction was performed by immunoprecipitation using a streptavidin resin to pull-down NTAP每GSK3汕 constructs. Full-length NTAP每GSK3汕 or its truncated constructs〞including deleted N-terminal NTAP每GSK3汕 constructs (amino acid 56ˋ433 and 353ˋ433), deleted C-terminal NTAP- GSK3汕 constructs (amino acid 1ˋ56 and 1ˋ353) and a kinase domain construct (amino acid 56ˋ353) were co-transfected with full-length Flag每LSD1 in 293 cells. Co-immunoprecipited HSP90, STIP1, and Flag每LSD1 were analyzed with Western blot using anti-HSP90, anti-STIP1 and anti-Flag antibodies, respectively. NTAP每GSK3汕 constructs were detected with an anti-calmodulin binding peptide (CBP) antibody. d The endogenous HSP90 complex in ARK2 cells were immunoprecipitated with an anti-HSP90 antibody in presence of scrambled siRNA or STIP1 siRNA. The proteins interacting with HSP90 (i.e., STIP1, LSD1, and GSK3汕) were identified with Western blot. e, f The interaction between Flag每LSD1 and NTAP每GSK3汕 in ARK2 cells was assayed by precipitation with streptavidin beads in presence of scrambled siRNA, STIP1 siRNA (e), or vehicle control (PBS) and Antp-TPR (20ˋ米M) (f). The STIP1, NTAP每GSK3汕, and Flag每LSD1 complexes were analyzed with Western blo
%U https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874249/