Anti tpo

ZY-444 was a gift from Professor Yihua Chen’s group at East China Normal University, Shanghai, China [17 (link)]. SCH772984 (ERK1/2 inhibitor, S7101) was purchased from Selleck, Shanghai, China. All drugs were dissolved in dimethyl sulfoxide solution.
The antibodies used in this study were: anti-PC (Santa Cruz Biotechnology, USA, sc-271493), anti-TSHR (Beyotime, AF1186), anti-NIS (Invitrogen, MA5-12308), anti-TPO (Abcam, UK, ab133322), anti-TG (Santa Cruz Biotechnology, sc-365997), anti-phospho-ERK1/2 (Cell Signaling Technology, USA, 4370), anti-ERK1/2 (Cell Signaling Technology,4695), anti-phospho-MEK1/2 (Cell Signaling Technology, 9154), anti-MEK1/2 (Cell Signaling Technology, 4694), anti-GAPDH (Beyotime, AF1186), and anti-α-tubulin (Beyotime, AF5012).

Antibodies used for immunoblot (IB), immunoprecipitation (IP), immunohistochemistry (IHC) and immunofluorescence (IF) were as follows. Anti-TBX3 antibodies for IB Anti-TBX3 (#ab99302, Abcam, 1:500). Anti-Flag (#3165, 1:1000), Anti-α-Tubulin (T5168, 1:15000) was purchased from Sigma. Anti-HA (#3724, 1:1500), Anti-Ubiquitin (#3936, 1:500), Anti-GST (#2622, 1:1000), was purchased from Cell Signaling Technology. Anti-USP15 (#A300-923A, BETHYL, 1:1000), Anti-USP15 (#67557-1-Ig, Proteintech 1:200), Anti-Ki-67 (#ab15580, Abcam, 1:100), Anti-Tpo (#ab278525, Abcam, 1:200), Anti-Tg (#ab156008, Abcam, 1:200), Anti-Nis (#MA5-12308, Thermo, 1:400), Anti-Gr-1 (#108401, BioLegend, 1:200), Anti-PAX8 (#ab53490, Abcam, 1:10), Anti-TTF1 (#ab76013, Abcam, 1:200) were employed according to the instructions. Reagents used in this study included MG132 (#S2619, Selleck), Puromycin (#P8230, Solario), CHX (#HY-12320, MCE), Blasticidin (#B9300, Solario), PLX4032 (#S1267, Selleck), SCH772984 (#S7101, Selleck), SR11302 (#E2813, Selleck).

Total cell extracts were obtained using RIPA buffer (Thermo Fisher Scientific, Waltham, MA USA) containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA; P8340). Equal amounts (25 µg) of Nthy-ori 3-1 and FTC-133 cell lysate proteins were separated under reduced conditions in 10% SDS-PAGE. Then the resolved proteins were electrotransferred onto a PVDF membrane (Millipore, Burlington, MA, USA) and incubated overnight in 2% BSA at 4 °C. The PVDF membranes were incubated for 1 h at RT with primary antibody anti-TPO (abcam, Cambridge, UK; ab203340) diluted 1:1000 in 2% BSA, and anti-GAPDH (Sigma-Aldrich, G9545) diluted 1:4000 in 2% BSA. After washing, the alkaline phosphatase (AP)-conjugated secondary antibody was used (diluted 1:4000): anti-mouse (Sigma-Aldrich, A2682) for TPO detection and anti-rabbit (Millipore, Burlington, MA, USA; AP304A) for GAPDH. The specific protein bands were visualized by colorimetric reaction after adding the substrates for AP: 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) (Roche, Mannheim, Germany). The relative protein expression was quantified densitometrically using ImageLab software (Bio-Rad, Hercules, CA, USA). The experiment was performed in duplicate.