Stress-induced phosphoprotein 1 acts as a scaffold protein for glycogen synthase kinase-3 beta-mediated phosphorylation of lysine-specific demethylase 1

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–LSD1 or HA-GSK3β in 293 cells and subsequently purified with Halo-tag resin. The interactions between LSD1 and GSK3β were examined with Western blot. b, c 293 cells were co-transfected with (1) FL flag–LSD1, (2) its deleted N-terminal Flag–LSD1 constructs (D1: amino acid 272？852, D2: amino acid 415？852, and D3: amino acid 515？852), (3) its deleted C-terminal Flag–LSD1 constructs (N1: amino acid 1？515, D2: amino acid 1？272), and (4) FL Halo-STIP1 or NTAP–GSK3β. After purification with a halo-tag resin (Halo-STIP1) or streptavidin beads (NTAP–GSK3β), co-immunoprecipitated Flag–LSD1 constructs were analyzed with Western blot using an anti-flag tag antibody. c The identification of GSK3β domains involved in the interaction was performed by immunoprecipitation using a streptavidin resin to pull-down NTAP–GSK3β constructs. Full-length NTAP–GSK3β or its truncated constructs—including deleted N-terminal NTAP–GSK3β constructs (amino acid 56？433 and 353？433), deleted C-terminal NTAP- GSK3β constructs (amino acid 1？56 and 1？353) and a kinase domain construct (amino acid 56？353) were co-transfected with full-length Flag–LSD1 in 293 cells. Co-immunoprecipited HSP90, STIP1, and Flag–LSD1 were analyzed with Western blot using anti-HSP90, anti-STIP1 and anti-Flag antibodies, respectively. NTAP–GSK3β 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 GSK3β) were identified with Western blot. e, f The interaction between Flag–LSD1 and NTAP–GSK3β 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 (20？μM) (f). The STIP1, NTAP–GSK3β, and Flag–LSD1 complexes were analyzed with Western blo