Sheep anti mouse igg na931

The anti-MRTF-A/B and anti-caldesmon antibodies were described previously [16 (link), 45 (link)]. Anti-tropomyosin (T2080), anti-α-actin (A5441), anti-vinculin (V9131), anti-Talin (T3287), anti-tubulin (T9024), and anti-FLAG (F7425) antibodies were from Sigma. Anti-SRF (sc-335), anti-zyxin (sc-6437), anti-Myc (sc-789), anti-RhoA (sc-418), anti-MYPT1 (sc-25618) and anti-c-Src (sc-18) antibodies were from Santa Cruz. Anti-pY118-phosphopaxillin antibody (#3818-1) was from Epitomics, anti-paxillin (MA3060) and anti-integrin αv (AB1930) antibodies from Chemicon, anti-GAPDH antibody (M171-3) from MBL, anti-HA antibody (3F10) from Roche, anti-integrin β1 antibody (610467) from BD Transduction Laboratories, and anti-FLAG antibody (018-22386) from Wako. Anti-FAK (#3285), anti-pY397-phosphoFAK (#3283), anti-pY576/577-phosphoFAK (#3281), anti-pY925-phosphoFAK (#3284), anti-pY527-phosphoSrc (#2105), anti-pY416-phosphoSrc family (#6943) and anti-pT696-phosphoMYPT1 (#5163) antibodies were from Cell Signaling Technology. The secondary antibodies, horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG (NA934) and sheep anti-mouse IgG (NA931) were from GE Healthcare, HRP-conjugated donkey anti-goat IgG (sc-2020) from Santa Cruz, and Alexa-conjugated goat anti-rabbit IgG (A-11034, A-11036) and goat anti-mouse (A-21049, A-11031) from Thermo Fisher Scientific.

Samples were thawed, resuspended in Laemmli Buffer (20% Glycerol, 4% SDS in 100 mM Tris Buffer, pH 6.8) and lysed in a Tissue homogenizer (Precellys Evolution, Bertin Instruments). BC Microstructure lysates were recovered, sedimented to remove cell debris, sonicated and stored at − 80 °C until use.
Protein quantification was performed in a Nanodrop ND-2000C (Thermo Scientific). Proteins were denatured and loaded in a electrophoresis gel (NuPAGE 4–12% Bis-Tris Gel) under reducing conditions for 50 min (200 V) and then electrophoretically transferred using a wet transfer system (Bio-Rad, 30 V, 18 h, 4 °C) into nitrocellulose membranes. Membranes were blocked for 1 h in TBS with 0.1% (w/v) Tween 20, 5% (w/v) non-fat dried milk and further incubated with the primary antibodies (Mouse anti-Human ERα, 1D5 Clone, Dako, final dilution 1:500; Rabbit anti-β tubulin, H-235, SC-9104, SantaCruz, final dilution 1:1000, used as loading control) and respective secondary HRP-conjugated secondary antibodies (Sheep anti Mouse IgG NA931; Donkey anti Rabbit IgG NA934; GE Healthcare, final dilution 1:20000). Membranes were developed using Amersham ECL Select Western Blot Detection Reagent (GE Healthcare) and visualized using a ChemiDoc System (BioRad).

Total cell extracts were harvested in RIPA buffer with protease inhibitor cocktail, PMSF and sodium orthovanadate (sc-24948, Santa Cruz). Cytoplasmic fractions were obtained using buffer A (10 mM HEPES pH 7.9, 1.5 mMgCl₂, 1 mM DTT) with 0.2% NP-40 and protease inhibitor cocktail; nuclear fractions were obtained using 1x PXL buffer (1x PBS without Mg²⁺/Ca²⁺, 0.1% SDS, 0.5% NP-40) with protease inhibitor cocktail. Immunoblotting was performed as described^{12 (link)}. Antibodies used are as follows: ESRP1 (27H12, mouse, 1:200)^{13 (link)}; FLAG (F1804, Sigma, mouse, 1:5000); β-tubulin (T8328, Sigma, mouse, 1:5000); mouse monoclonal U2AF65 (kindly provided by Juan Valcarcel, 1:5000). Secondary antibody was purchased from GE Healthcare Life Sciences (sheep anti-Mouse IgG NA931 from 1:2000 to 1:10000).