![Phosphorylation of phospholamban (PLN) monomers in heart tissue is reduced if PLN can form pentamers. (A, B) Western blot using antibodies against PLN phosphorylated at S16 [anti-pPLN(S16)], T17 [anti-pPLN(T17)], and total PLN (anti-PLN); heart tissue was quickly harvested from mice after anaesthesia for 20 min; for comparison of monomer phosphorylation under equal conditions, total protein loads were lower for TgAFA-PLN (2.2-fold) to match the amounts of total PLN monomers in SDS gels; m, monomers (∼6 kDa); p, pentamers (∼25 kDa); bar graph shows proportion of S16-(T17-)phosphorylated PLN monomers (pPLN(S16/T17)m) relative to total PLN monomers (PLNm); N = 6–7 hearts per group. (C, D) Western blot using Phos-tag™-gels; graphs show percentage of phosphorylated PLN monomers (pPLNm); (C) heart lysates as in A and anti-PLN antibodies; N = 7 hearts per group; (D) heart lysates of independent mouse lines expressing PLN and AFA-PLN transgenes tagged with a FLAG epitope at the N-termini (TgF-PLN and TgF-AFA-PLN); tagged protein was detected by anti-FLAG antibodies; mx, px, monomers (pentamers) with x = 0–10 phosphate group(s); groups were compared by unpaired two-tailed Student’s t test.](https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/cardiovascres/119/7/10.1093_cvr_cvad037/1/cvad037f2.jpeg?Expires=1749524540&Signature=wVghgqVtpabPDr~92pqp7LjQU~CLpVknMeE13LulPcb8S3CRzkp2mD2XVIp86ig7s6vBHt4a0EKJaMu05Sq2P-1fMhZdUfsx2QoBRpgNu83-gPIFc0~P3fn4od0ibz1W1JDdOmmNouk3aVQdRS6XgK4G9qRNDzMPIGA6-mG61PoxcDveDv2PUzYhhlsfNTy1VGP9oOfQBQT1X2EdxFoI4nKUoUjaUHO20hpQxJ-q~rfk~Ch4verbcPx2VYBq1h4pyrdJevtucMgH4AOiGZ0kRoQeqGFLJnldqviWSdi3ydr5WtAlRIpLnQaRJJWlZoqfAChah-SVM72vK8P4hDSAYQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Phosphorylation of phospholamban (PLN) monomers in heart tissue is reduced if PLN can form pentamers. (A, B) Western blot using antibodies against PLN phosphorylated at S16 [anti-pPLN(S16)], T17 [anti-pPLN(T17)], and total PLN (anti-PLN); heart tissue was quickly harvested from mice after anaesthesia for 20 min; for comparison of monomer phosphorylation under equal conditions, total protein loads were lower for TgAFA-PLN (2.2-fold) to match the amounts of total PLN monomers in SDS gels; m, monomers (∼6 kDa); p, pentamers (∼25 kDa); bar graph shows proportion of S16-(T17-)phosphorylated PLN monomers (pPLN(S16/T17)m) relative to total PLN monomers (PLNm); N = 6–7 hearts per group. (C, D) Western blot using Phos-tag™-gels; graphs show percentage of phosphorylated PLN monomers (pPLNm); (C) heart lysates as in A and anti-PLN antibodies; N = 7 hearts per group; (D) heart lysates of independent mouse lines expressing PLN and AFA-PLN transgenes tagged with a FLAG epitope at the N-termini (TgF-PLN and TgF-AFA-PLN); tagged protein was detected by anti-FLAG antibodies; mx, px, monomers (pentamers) with x = 0–10 phosphate group(s); groups were compared by unpaired two-tailed Student’s t test.
