Anti timp2

Experiments were performed as previously described [14 (link)]. If not declared otherwise, 3 days before harvesting, cells were transfected with siRNA as indicated. The following antibodies were used: anti-KMT9α (#27630, lot 20062017, Schüle Lab), anti-KMT9β (#28358, lot 27022018, Schüle Lab), anti-H4K12me1 (#27429, lot 27062017, Schüle Lab); anti-H4 (ab31830, lot GR3204774-2, abcam); anti-Tubulin (alpha tubulin, #T6074, lot 03714804 V, Sigma), anti-LMNA (sc-20680, lot F2607, Santa Cruz); anti-GAPDH (MAB574, lot 3273148, R&D systems); anti-SMARCA2 (NB100-55308; lot A1; Novus biologicals); anti-TIMP2 (CST#5738, lot 3, Cell signaling); anti-SOD2 (CST#13194, lot 1, Cell signaling); anti-YES1 (#PA5-80243, lot VA2919193, Invitrogen). Proteins from patient tissues were extracted using the Minilys homogenizer (Bertin instruments) and RIPA buffer (1 mM EDTA, 50 mM Tris–HCL pH7.5, SDS 0.1%, NaCl 150 mM, NP-40 1%, Na deoxycholate 1%, protease inhibitor cocktail EDTA-free). Samples were cycled for 15 s at top speed.

Fetal bovine serum (FBS), penicillin and streptomycin were ordered from Gibco. Baicalein, SB203580 and anisomycin were ordered from Sigma. Dulbecco's modified Eagle's medium (DMEM) was purchased from Invitrogen. Anti-p38, anti-Phospho-p38 (Thr180/Tyr182), anti-MMP-2, anti-MMP-9, anti-TIMP-1 and anti-TIMP-2 antibodies were purchased from Cell Signaling. Anti-β-actin was purchased from Santa Cruz.

Protein lysates were prepared using RIPA lysis buffer (Thermo Fisher Scientific), and protein concentration was determined by BCA Protein Assay Kit (Thermo Fisher Scientific). Protein lysates were separated by SDS-PAGE and transferred onto PVDF membrane (Bio-Rad). After blocking, the blot was incubated with primary antibody at 4 °C overnight, followed by incubation with secondary antibody. Chemiluminescence signal was visualized using SuperSignal Pico PLUS chemiluminescent Substrate (Thermo Fisher Scientific). The following primary antibodies were used in this study: anti-GM130 (1:1000, #12480); anti-CD54 (1:1000, #4915); anti-Annexin V (1:1000, #8555); anti-CD9 (1:1000, #13174); anti-TIMP2 (1:1000, #5738); anti-TIMP3 (1:1000, #5673); anti-GAPDH (1:1000, #5174); anti-VEGFR2 (1:1000, #9698); anti-ERK (1:1000, #4695); anti-p-ERK (1:1000, #4370); anti-MMP2 (1:1000, #87809); anti-MT1-MMP (1:1000, #13130) antibodies were purchased from Cell Signaling technologies (Beverly, MA, USA).