Pakt s473 9271

Chemicals were purchased from Sigma‐Aldrich unless otherwise stated. Cell culture supplies were purchased from Life Technologies. Endothelin 1 (CSCSSLMDKECVYFCHLDIIW) was purchased from Bachem (Product No. 4040254) and is referred to as EDN for the purpose of all stimulation experiments. The following key reagents were employed in the study: TiO₂ (GL Sciences), ¹³C₆‐¹⁵N₂ lysine (Lys‐8, Silantes GmbH) and ¹³C₆‐¹⁵N₄ arginine (Arg‐10, Silantes GmbH). Heavy peptides for SRM were synthesised by JPT Peptide Technologies. Kinase inhibitors were purchased from Selleckchem, except for ddcAMP (Enzo Life Sciences), GSK‐429286, CRT0066101 and JNK‐IN‐8 (Sigma‐Aldrich). Primary antibodies and anti‐rabbit secondary antibodies (7074) were purchased from Cell Signalling Technologies [pAKT S473 (9271), pCREB S133 (9198), PKC motif (6967), PKA motif (9624), pS6 S235/6 (4858), pRB S807/11 (8516), CaMKII S286 (12716), pMAPKAPK2 T222 (3316), p‐cJun S73 (3270), p‐cRAF S338 (9427), pACC S79 (11818), pHSP27 S82 (9709), AKT (4691) and CREB (9197)]. Antibodies against β‐Actin (AC‐15), GAPDH (MAB374) and EDNRB (21,196) were purchased from Sigma‐Aldrich, Millipore and Santa Cruz, respectively. Anti‐mouse and anti‐goat secondary antibodies were purchased from Jackson ImmunoResearch.

Cells or tissues were homogenized in an l-RIPA lysis buffer (R0020, Solarbio). Protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, immunoblotted with the indicated antibodies, and visualized using the ECL. The antibody information and dilutions were as follows: METTL3(D2160,Cell Signaling Technology),1:3000; Akt(9272, Cell Signaling Technology),1:5000; p-Akt (S473)(9271, Cell Signaling Technology),1:5000; UCP1(U6382, Sigma),1:5000; PGC-1a(66369-1-lg, Proteintech),1:1000; β-Actin(60008-1-lg, Proteintech),1:5000; PPARg(16643-1-AP, Proteintech),1:300 0; GAPDH(60004-1-lg, Proteintech),1:5000; Total OXPHOS (ab110413, Abcom),1:2000; PRDM16(A11581, ABclonal),1:1000; YTHDF1(17479-1-AP, proteintech),1:2000; YTHDF2(24744-1-AP, proteintech),1:5000; YTHDF3(25537-1-AP, proteintech),1:3000.