Sds 4 15 polyacrylamide gels

A total of 100 nM of Neratinib was added to 5 × 10⁵ cells for 24 h and 10 μg each of protein lysates were separated on SDS/4–15% polyacrylamide gels (Bio-Rad) and transferred onto nitrocellulose membranes (Invitrogen). The blots were incubated with antibodies directed against GAPDH [(14C10) cell signaling #2218], HER2 [(29D8) cell signaling #2165], Erk1/2 [cell signaling #9102], Phospho-Erk1/2 [(Thr202/Tyr204) cell signaling #9101], phospho-S6 ribosomal protein [(Ser235/236) cell signaling #2211], and S6 ribosomal protein [(5G10) cell signaling #2217] and with the relevant secondary antibodies and visualized using Odyssey Clx (LI-COR). All blots were derived from the same experiment and were processed in parallel. In Supplementary Material, all blots and gels are accompanied by the locations of molecular weight/size markers and uncropped scans (Supplementary Fig. 2).

Proteins were isolated from 2 to 5×10⁵ cells using RIPA buffer (BP-115X) supplemented with the HaltTM Protease inhibitor cocktail (Thermo Scientific 78425). Protein lysates (10μg each) were separated on SDS/4-15% polyacrylamide gels (Bio-Rad) and transferred to nitrocellulose membranes (Invitrogen). The blots were incubated with antibodies directed against GAPDH (sc-47724, mouse monoclonal, 1:1000), Estrogen Receptor α (D8H8) (Cell signaling-8644, Rabbit monoclonal, 1:500) and with the relevant secondary antibodies which were HRP conjugated (Anti-rabbit IgG, HRP-linked Antibody cell signaling-7074, Anti-mouse IgG, HRP-linked Antibody Cell signaling-7076) and visualized using Clarity Western ECL Substrate (BIORAD) and G box (Syngene).