Anti ha matrix

HopW1-HA was immunoprecipitated with anti-HA matrix (Roche) from transgenic Arabidopsis or transiently transformed N. benthamiana as described Text S1. LC-MS/MS protein identification after trypsin digestion of protein bands was performed at Chicago Biomedical Consortium as described [47] (link); data was searched against the NCBI database using Mascot and validated with Scaffold 2 (Proteome Software Inc.)

Activated CD4 cells (as mentioned above) were transfected with Casp8p41-HA expression vector using Lanza Nucleofector IIb system. One hour after electroporation, cells were treated with Z-VAD-fmk (10 μM) and Ixazomib (100 nM) for 4 h (to prevent protein degradation and cell death induced by Casp8p41 expression), followed by treatment with or without bryostatin (10 ng/ml) for an additional 1 h. Cells were collected and washed and lysis with cell lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1.0% Tween-20, protease and phosphatases inhibitors). Five hundred micrograms of total cell lysate were diluted in 500 μl of buffer, precleared with 25 μl Protein A/G agarose (Santa Cruz). Precleared lysate incubated with Anti-HA matrix (cat # 11815016001, Roche, Germany) or 2 μg anti–BCL2 (SC-509, Santa Cruz) or mouse IgG for overnight at 4C. After incubation, 20 μl of Protein A/G agarose was added to the appropriated samples, and complexes were washed five times in IP buffer (20 mM Tris, pH 7.5, 300 mM NaCl, 1.0% Tween-20, protease and phosphatases inhibitors) and boiled in 20 μl 2× SDS-PAGE loading buffer, subjected to SDS-PAGE, and immunoblotted with HA-HRP antibody (Roche) or BCL2 antibody.

IP protocol was previously described^{93 (link)}. Protein lysates (1 mg per 1 ml) were suspended in 1 ml of lysis buffer containing a protease inhibitor cocktail and incubated with 20 µL anti-Flag matrix (Sigma, A2220) or anti-HA matrix (Roche, 11815016001) overnight at 4 °C with end-over-end rotation. Unbound proteins were removed by washing 4 times with 1 ml of lysis buffer and with spinning at 2500 rcf for 1 min each time. Precipitated proteins were denatured in 2× reducing SDS-PAGE sample buffer and boiled for 5 min at 95 °C.