Lyn and PAG are associated in B-NHL rafts. (A) Raft (R) and nonraft (NR) aliquots (100 mL) from Raji, DoHH2, SUDHL6, Val, OCI-Ly3, Granta-519, Jeko-1, SR786, and L428 cells containing each equivalent amounts of either PAG or Lyn were IP with Sepharose-4B-coupled anti-PAG C-1 antibody (IP PAG, top 2 rows), or protein A/G-bound anti-Lyn antibody (IP Lyn, bottom 2 rows). IP materials were probed with anti-PAG MEM-255 mAb (first and third rows) or anti-Lyn antibody (second and fourth rows). Open rectangles indicate the H chain (HC) of the antibody used for IP. Vertical lines indicate repositioned gel lanes. (B) R and NR material (100 μL) from Raji, DoHH2, and SUDHL6, as in panel A) was IP with anti-PAG C-1 bound to Sepharose, and probed with anti-Csk antibody. Total: R and NR material (10 μL) probed directly with anti-Csk. (C) R material from DoHH2 (100 μL) was IP with Sepharose-bound anti-PAG C-1 (IP PAG) in 3 aliquots and probed by WB with: top, anti-Lyn; middle, anti-pY418 Src; and bottom, anti-pY505 Lck. Total: R material (10 μL) probed directly with the indicated antibodies. (D) PNS of 15 × 106 DoHH2 and SUDHL6 cells extracted with different detergents, subjected to IP with anti-PAG (PAG C-1) and WB with anti-PAG MEM 255, anti-Lyn, and anti-Src pY418. (E) R and NR material (100 μL) from SUDHL6 was IP with protein A/G-bound anti-Lyn, or anti-pY505 Lck antibodies, and probed with anti-PAG MEM-255 (first row) and anti-Lyn (second row). IPs described in this figure were carried out at least 3 times for each cell line.
