Anti grb2

Rabbit polyclonal antibodies to ERK (ERK1/ERK2) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); anti-phospho-ERK protein, anti-MEK (MEK1/MEK2), and anti-p-MEK1/2 were purchased from Cell Signaling Technology (Beverly, MA), and anti-tubulin was from Covance (Princeton, NJ). Monoclonal antibodies Anti-C-RAF, anti-pan-Ras, and anti-Grb2 were obtained from BD Transduction Laboratories (Franklin Lakes, NJ), whereas rabbit polyclonal anti-Shoc2 was produced and purified in the lab. Anti-HA and AU5 monoclonal antibodies (mAb) were purchased from Berkeley Antibody Company (Berkeley, CA). Anti β-actin mAb, and recombinant human basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and epidermal growth factor (EGF) were from Sigma-Aldrich (St. Louis, MO). Anti-mouse and rabbit HRP-conjugated mAb were from GE Healthcare Bio-Sciences (Piscataway, NJ).

Transient transfection of A36-p14 hybrid constructs, CB6-Src-GFP, pE/L-GFP-Nck (SH2) and the pE/L-LifeAct-iRFP670 expression in Vaccinia-infected cells was done using FUGENE (Promega) as described by the manufacturer. To transiently express the A36-p14 chimera and its variants (Figure 3B-E), expression vectors containing the relevant construct under the control of the A36 promoter were transfected into cells one hour after infection with the WR-ΔA36 virus (Parkinson and Smith, 1994 (link)). pE/L-LifeAct-iRFP670 (Figures 4A and 6A) and pE/L-GFP-Nck(SH2) (Figure 6B) were transfected into cells one hour after infection with the relevant viruses. CB6-Src-GFP (Figure 6A) was transfected into cells 16 hr prior to infection. For knockdown experiments, HeLa cells were transfected with siRNA as previously described (Abella et al., 2016 (link)). Cells were infected with Vaccinia virus 72 hr after siRNA transfection, and samples from each siRNA condition were kept for immunoblot analysis. The following siRNAs were used: AllStars (Qiagen; SI03650318), Grb2-targeting siRNA oligos AGGCCGAGCGUAAUGGUAA, GAAAGGAGCUUGCCACGGGUU and CGAAGAAUGUGAUCAGAACUU. The following primary antibodies were used in immunoblots: anti-vinculin (Sigma #V4505; 1:2000), anti-Grb2 (BD Transduction #610112, 1:3000). HRP-conjugated secondary antibodies were purchased from The Jackson Laboratory.