Anti actin a2228

Cells were grown to 60-70% confluence and lysed with standard RIPA buffer. Frozen xenograft tumors were finely grinded using cold hammer and a pestle. The pestle was placed over dry ice to maintain it at low temperatures. The ground tumor was suspended in ice-cold PBS and dounce homogenized and subsequently washed twice with ice-cold PBS. The cell pellets obtained from washed xenografts were processed like the pellets obtained from in-vitro experiments. The resulting total cell lysate was run on SDS-PAGE gel, transferred on to nitrocellulose membrane and immunoblotted using antibodies for the proteins of interest. Antibodies used for immunoblotting are anti-ER (HC20 from Santa Cruz), anti-PR KD68 (in-house developed) [65 (link)], anti-actin (A-2228 from Sigma). KD68 picks up both the PR isoforms in immunoblots as well as immunoprecipitation [1 (link)]. Protein expression was normalized to the actin loading control.

The WT-STK39, KR-STK39 and CA-STK39 were from Jim McCormick (OHSU) and James Wohlschlegel (UCLA). Plasmids of wild-type and deletion mutants for SNAI1 were generated as described 32 (link); all sequences were verified by DNA sequencing. Antibodies purchased from Sigma-Aldrich (St. Louis, MO) include: anti-Flag (F3165), 1:4000; anti-Actin (A2228), 1:10000; anti-Myc (9E10), 1:3000. Anti-STK39 (2281), 1:1000; anti-SNAI1 (3879), 1:1000; and α-Tubulin (2144), 1:1000 were from Cell Signaling (Danvers, MA). N-cadherin (05-915), 1:1000 was from Upstate (Lake Placid, NY). Anti-HA (3F10), 1:10000 was from Roche (Madison, WI) and anti-CDH1 (610181), 1:1000 was from BD Bioscience (San Jose, CA). Anti-Lamin A/C (sc-376248), 1:1000 was from Santa Cruz (Dallas, TX). The p-T203-SNAI1 antibody was from Dr. Greg Longmore. STK39 shRNA expression plasmids were purchased from MISSION shRNA at Sigma-Aldrich. TGFβ1 was from Peprotech. STOCK2S 26016 (STO) was from Tocirs (Minneapolis, MN) and MG132 was from Sigma.