Expression levels of BTLA, CD28, HVEM, and PD-L1 on Jurkat or Raji cells. (A) Flow cytometry histograms of BTLA surface expression in parental Jurkat cells. (B) Flow cytometry histograms of CD28 surface expressions in the indicated types of Jurkat cells. (C) Quantification of BTLA-mGFP and PD-1–mGFP in Jurkat (BTLA-mGFP⁺) and Jurkat (PD-1–mGFP⁺) cells, respectively. 4 × 10⁶ Jurkat (BTLA-mGFP⁺) cells or Jurkat (PD-1–mGFP⁺) cells were lysed on ice using PBS containing 1% NP-40 and protease inhibitors. BTLA-mGFP and PD-1–mGFP were then immunoprecipitated using GFP-Trap and eluted by 50 µl SDS sample buffer. 10 µl and 5 µl of the eluates were loaded to SDS-PAGE together with indicated amounts of purified mGFP protein as standards (Stds). BTLA-mGFP and PD-1–mGFP were then quantified using a standard curve constructed based on the mGFP standards and their concentrations in Jurkat cells calculated by dividing the number of moles by the average volume of a Jurkat cell, assuming a 12-µm diameter for Jurkat cells. (D) Red histogram indicates HVEM surface expression in Raji (HVEM-mRuby⁺) cells. Blue histogram indicates PD-L1 surface expression on Raji (PD-L1–mCherry⁺) cells. Gray and black histograms show isotype control staining.