Fig 1: TRIM44 facilitates SQSTM1 S349 phosphorylation via PKA(A) U266 TRIM44[OE-CON], TRIM44[OE] cells were exposed to inhibitors targeting CKI (CKI-7, 100 μM, 6 h), GSK3 (CHIR-99021, 2 μM, 24 h), CaMKII (KN-62, 1 μM, 24 h), and PKA (H89, 5 μM, 24 h), and As[III] (5 μM, 6 h) or left untreated. Whole-cell lysates were examined using anti-SQSTM1, anti-SQSTM1 p-S349. GAPDH served as a loading control.(B) Cell extracts from U266 TRIM44[OE-CON], TRIM44[OE], TRIM44[KD-CON] and TRIM44[KD] cells were lysed for measure PKA activity (PKA Kinase activity Kit, Abcam, ab139435). Measurements were made in triplicates, mean ± SD. Student’s ttest was used for statistical analysis *P < 0.05, **P < 0.01.(C) 293T cells stably co-expressing FLAG-HA-SQSTM1 with TRIM44 (TRIM44[OE]) or without TRIM44 (TRIM44[OE-CON) were treated with or without As[III] (5 μM, 6 h). Immunoprecipitation was subsequently performed with an anti-PKA substrate antibody and immunoblotting for indicated signals.(D) U266 TRIM44[KD-CON], TRIM44[KD] cells were treated with or without As[III] (5 μM, 24 h). Whole-cell lysates were analyzed via immunoblotting for SQSTM1 p-S349, SQSTM1, PKA Cα.(E) U266 TRIM44[OE-CON] and TRIM44[OE] cells were transfected with NC or PKA Cα siRNA. After 48 h, whole-cell lysates were analyzed via immunoblotting for SQSTM1 p-S349 and SQSTM1, PKA Cα.(F) 293T cells stably co-expressing FLAG-HA-SQSTM1 with TRIM44 (TRIM44[OE]) or without TRIM44 (TRIM44[OE-CON) were treated with or without Baf A1 (100 nM, 6 h). Immunoprecipitation was subsequently performed with an anti-HA antibody and immunoblotting for indicated signals.
Fig 2: TRIM44 facilitates SQSTM1 S349 phosphorylation via PKA. (A) U266 TRIM44[OE-CON], TRIM44[OE] cells were exposed to inhibitors targeting CKI (CKI-7, 100 µM, 6 h), GSK3 (CHIR-99021, 2 µM, 24 h), CaMKII (KN-62, 1 µM, 24 h), and PKA (H89, 5 µM, 24 h), and As[III] (5 µM, 6 h) or left untreated. Whole-cell lysates were examined using anti-SQSTM1, anti-SQSTM1 p-S349. GAPDH served as a loading control. (B) Cell extracts from U266 TRIM44[OE-CON], TRIM44[OE], TRIM44[KD-CON] and TRIM44[KD] cells were lysed for measure PKA activity (PKA Kinase activity Kit, Abcam, ab139435). Measurements were made in triplicates, mean ± SD. Student's t test was used for statistical analysis *P < 0.05, **P < 0.01. (C) 293T cells stably co-expressing FLAG-HA-SQSTM1 with TRIM44 (TRIM44[OE]) or without TRIM44 (TRIM44[OE-CON) were treated with or without As[III] (5 μM, 6 h). Immunoprecipitation was subsequently performed with an anti-PKA substrate antibody and immunoblotting for indicated signals. (D) U266 TRIM44[KD-CON], TRIM44[KD] cells were treated with or without As[III] (5 μM, 24 h). Whole-cell lysates were analyzed via immunoblotting for SQSTM1 p-S349, SQSTM1, PKA Cα. (E) U266 TRIM44[OE-CON] and TRIM44[OE] cells were transfected with NC or PKA Cα siRNA. After 48 h, whole-cell lysates were analyzed via immunoblotting for SQSTM1 pS349 and SQSTM1, PKA Cα. (F) 293T cells stably co-expressing FLAG-HA-SQSTM1 with TRIM44 (TRIM44[OE]) or without TRIM44 (TRIM44[OE-CON) were treated with or without Baf A1 (100 nM, 6 h). Immunoprecipitation was subsequently performed with an anti-HA antibody and immunoblotting for indicated signals.
Supplier Page from Abcam for PKA Kinase Activity Assay Kit