![Figure 1. CD28 preferentially associates with p85β. (A) Ratio of p85α/p85β molecules per nanogram of total mRNA determined by QRT-PCR. (B) Jurkat cells transfected with control, rHAp85α, or rHAp85β vectors (36 hours), were activated via CD3+CD28 (15 minutes). WB examined protein expression in total extracts, extracts were also immunoprecipitated with anti-CD28 Ab, and associated p85 was analyzed in Western blot (WB) using anti-HA Ab (long and short exposure). (C) Number of p85α or p85β molecules per nanogram of total mRNA (from p85β−/− and p85β+/+ T cells) by QRT-PCR; each dot represents a mouse. Western blot analysis of p85 in extracts of a representative mouse (vertical line indicate a repositioned gel lane). (D) T cells from p85β−/− and p85β+/+ mice and Jurkat cells were activated as in panel A. Total extracts or CD28 immunoprecipitates were resolved in 2-dimensional electrophoresis and analyzed in WB using anti–pan-p85 antibody. β indicates the p85β spot (absent in p85β−/− mice). The isoelectric point (pI) of mp85β was 5.74, and the pIs were 5.96, 5.9, and 5.8 for mp85α and its phosphorylated forms; the pI for hp85β was 6.03, and the pIs were 5.84, 5.77, and 5.71 for hp85α and its phosphorylated forms. Graphs show the mean plus or minus SD (n = 3) of the β spot signal relative to the combined signal for all spots (100%). (E) Jurkat cells were activated as in panel B; WB shows the amount of p85 present in CD3 or CD28 immunoprecipitates. Graphs show the mean signal plus or minus SD (AU indicates arbitrary unit) of p85 bound to CD3 or CD28. (F) cDNA encoding human (h) or mouse rp85α or rp85β was transcribed/translated in vitro (rhp85α was fused to GFP) in the presence of 35S-Met. Purified proteins were examined by SDS-PAGE and autoradiography (top left). Sequence of the hCD28 peptides; the dots represent the nonconserved amino acids in mCD28 (H > P and P > A). In the bottom, p85 bound to the different CD28 peptide columns analyzed by SDS-PAGE and autoradiography. PDGFR (P) peptide was used as a positive control; column with no peptide and mp85α was used as the negative control. The histograms show the absolute binding of each rp85 form to CD28 motifs (mean signal ± SD in arbitrary units [AU]) on the right, and rp85 relative binding to M peptides and to P column (n = 3). *t test, P < .05.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/113/14/10.1182_blood-2008-04-152942/4/zh80170934080001.jpeg?Expires=1735815307&Signature=dd-AipUm1VS0QkGP-kwpd4t6z-sLJ1XIo5X1aNLF-zRkH9Qk~daT4O38Uc6bVfVjPonNbiKXc7pH-j8VK7kH8hYI2x9khC4FpLuU8zdyiM0RMxtlzBPj-kiaWqxxk176se9Ffq5xYXJjd4eEb-aHGQZRtF64vopAve5nGol3pQNqty1i-AvljX~552C8NKSUWJL2AHS1VPICHA1DMHvZEhpK7iOz6uGtri1w1CAikkB0bbfhKbD045kwxKSrOEHBuVdYMcHz3WuG7u3qMLv-KMIucH8eak1YssSbIjoCHhbNyJf9wIUgQoskKioPJs4170xLgDeULlA1e3~QShWJtA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
CD28 preferentially associates with p85β. (A) Ratio of p85α/p85β molecules per nanogram of total mRNA determined by QRT-PCR. (B) Jurkat cells transfected with control, rHAp85α, or rHAp85β vectors (36 hours), were activated via CD3+CD28 (15 minutes). WB examined protein expression in total extracts, extracts were also immunoprecipitated with anti-CD28 Ab, and associated p85 was analyzed in Western blot (WB) using anti-HA Ab (long and short exposure). (C) Number of p85α or p85β molecules per nanogram of total mRNA (from p85β−/− and p85β+/+ T cells) by QRT-PCR; each dot represents a mouse. Western blot analysis of p85 in extracts of a representative mouse (vertical line indicate a repositioned gel lane). (D) T cells from p85β−/− and p85β+/+ mice and Jurkat cells were activated as in panel A. Total extracts or CD28 immunoprecipitates were resolved in 2-dimensional electrophoresis and analyzed in WB using anti–pan-p85 antibody. β indicates the p85β spot (absent in p85β−/− mice). The isoelectric point (pI) of mp85β was 5.74, and the pIs were 5.96, 5.9, and 5.8 for mp85α and its phosphorylated forms; the pI for hp85β was 6.03, and the pIs were 5.84, 5.77, and 5.71 for hp85α and its phosphorylated forms. Graphs show the mean plus or minus SD (n = 3) of the β spot signal relative to the combined signal for all spots (100%). (E) Jurkat cells were activated as in panel B; WB shows the amount of p85 present in CD3 or CD28 immunoprecipitates. Graphs show the mean signal plus or minus SD (AU indicates arbitrary unit) of p85 bound to CD3 or CD28. (F) cDNA encoding human (h) or mouse rp85α or rp85β was transcribed/translated in vitro (rhp85α was fused to GFP) in the presence of 35S-Met. Purified proteins were examined by SDS-PAGE and autoradiography (top left). Sequence of the hCD28 peptides; the dots represent the nonconserved amino acids in mCD28 (H > P and P > A). In the bottom, p85 bound to the different CD28 peptide columns analyzed by SDS-PAGE and autoradiography. PDGFR (P) peptide was used as a positive control; column with no peptide and mp85α was used as the negative control. The histograms show the absolute binding of each rp85 form to CD28 motifs (mean signal ± SD in arbitrary units [AU]) on the right, and rp85 relative binding to M peptides and to P column (n = 3). *t test, P < .05.
