Western blot analysis of Ubiquitin-protein conjugates, pAb: Lane 1: MW marker, Lane 2: Poly-ubiquitin chains (Ub2-7) (K48-linked)

Overlay histogram showing Hela cells stained with SH2B3(green line). The cells were fixed with 2% paraformaldehyde (10 min) and then permeabilized with 90% methanol for 10 min. The cells were then icubated in 2% bovine serum albumin to block non-specific protein-protein interactions followed by the antibody (SH2B3, 1:25 dilution) for 60 min at 37ºC. The secondary antibody used was Goat-Anti-Rabbit IgG, DyLight® 488 Conjugated Highly Cross-Adsorbed(OE188374) at 1/200 dilution for 40 min at 37ºC. Isotype control antibody (blue line) was rabbit IgG1 (1ug/1x10^6 cells) used under the same conditions. Acquisition of >10,000 events was performed.