Mouse anti gapdh ab125247

5 mL of 0.6 O.D.₆₀₀ of exponentially growing cultures were harvested by centrifugation and cell pellets were boiled for 5 min in 250 μL of ACB (25 mM Tris-HCl (pH 6.8), 10% glycerol, 5% 2-mercaptoethanol, 2% SDS, 8 M urea). Proteins were immunostained using the indicated primary antibodies (Rat anti-HA 3F10, Roche, Basel, Switzerland; mouse anti-Myc 9E10 631206, Clontech, Mountain View, California, USA; mouse anti-p73 5B1288, Novus Biologicals, Littleton, Colorado, USA; mouse anti-GAPDH ab125247, Abcam, Cambridge, UK; rabbit anti-Arp5 ab12099, Abcam) and HRP-conjugated secondary antibodies (rabbit anti-mouse, Dako, Glostrup, Denmark; swine anti-rabbit, Dako) as instructed by manufacturers.

100 mL of 0.6 O.D.₆₀₀ of exponentially growing cultures were suspended in 500 μL of IP lysis buffer (Pierce Biotechnology/Thermofisher, Waltham, Massachusetts, USA) containing 1X cOmplete (Roche) and 1 mM Pefabloc (Sigma-Aldrich, St. Louis, Missouri, USA). After addition of 500 μL of glass beads, samples were broken as previously described [72 (link)] and then disrupted on a glass-bead beater (MM400 Retsch, Haan, Germany) at 25 Hz at 4°C. 100 μL of 20% protein G-Sepharose fast flow (Sigma-Aldrich) were washed in wash buffer (PBS 1×/0.2% Igepal) then suspended in 500 μL washing buffer. Protein G-Sepharose were mixed with 1.5 μg of rat anti-HA 3F10 for 2h at 4°C. Beads were washed 3 times with washing buffer and incubated with 1 mg of total protein overnight at 4°C. Beads were then washed 5 times with washing buffer and boiled with 65 μL of ACB. Samples were analyzed by western blotting using the indicated primary (Rabbit anti-HA 631207, Clontech; mouse anti-Myc 9E10, Clontech; mouse anti-GAPDH ab125247, Abcam) and secondary antibodies (swine anti-rabbit, Dako; anti-mouse Veriblot, Abcam).