Figure 2

Absolute quantification of endogenous proteins in HEK293T cells. (A) Top: Different amounts of recombinant GFP were loaded on a polyacrylamide gel and visualized by Western blot analysis using a GFP-specific antibody. Bottom: Linear regression model analysis of the recombinant GFP signals. (B) Top: HEK293T cells were transfected with an empty vector as a control or a plasmid encoding GFP-tagged PI4KIII β and cultured for 24 h. Cells were lysed, and expression of endogenous and ectopically expressed PI4KIII β was detected by Western blot analysis using a PI4KIII β-specific antibody. Detection of tubulin served as a loading control. Bottom: Different amounts of recombinant GST-tagged PI4KIII β were loaded on a polyacrylamide gel and visualized by Western blot analysis using a PI4KIII β-specific antibody. (C) Comparison of absolute quantifications via GST-PI4KIII β standard (blue) and GFP-standard (black).