2.7. Phospho‐protein analysis

To determine the effect of ZYBT1 on phosphorylation of BTK and PLCγ2, THP‐1 cells were treated with increasing concentration of ZYBT1 for 1 hour followed by a brief pervanadate (500 µmol/L final concentration) treatment for 10 minutes in 96 well plates. The cells were washed thrice with ice cold sterile PBS, lysed in SDS‐PAGE loading dye and analyzed by western blot using human anti‐phospho‐BTK (Y223) antibody or human phospho‐PLCγ2 (Y1217) antibody. For total cellular BTK and PLCγ2, blots were stripped and re‐probed with human anti‐BTK antibody or human anti‐PLCγ2 antibody. To determine receptor resident time, T₀ and T₂₄ sets were prepared as described above, only with the exception that in this case no labeled compound were added and cells were lysed after the stipulated time and analyzed in SDS‐PAGE and immunoblotting.