Extended Data Fig. 1: Proteasomal degradation of PAK4 by PKA activation.
From: p21-activated kinase 4 counteracts PKA-dependent lipolysis by phosphorylating FABP4 and HSL
a. 3T3-L1 adipocytes were transfected with catalytic subunit of PKA (His-PKA Cα) in combination with PAK4 on day 4 post-MDI treatment. Seven days after MDI treatment, PKA binding with PAK4 in the presence of 3 μM MG132 was analysed by Co-IP assay. b. Recombinant PAK4 was incubated with recombinant PKA and [32P]ATP for 30 min, and the proteins in the mixture were resolved by SDS-PAGE. The band was visualized by autoradiography of 32P-labelled protein. Protein loading was confirmed by Coomassie blue staining. c. 3T3-L1 adipocytes transfected with PKA and PAK4 were treated with isoproterenol (1 μM) for 1 h. Phosphorylation of PAK4 was determined in PAK4 immunoprecipitates using LC-MS/MS analysis after in-gel digestion. Representative spectra are shown. d. 3T3-L1 adipocytes were transfected with siRNA targeting various E3 ubiquitin ligases, followed by treatment with isoproterenol (1 μM) for 1 h. Protein levels of PAK4 were analyzed by Western blotting (left), while mRNA levels were assessed using qPCR (n = 7). Data are representative of at least three independent experiments. Values are mean ± SD. Unpaired two-tailed t-test between two groups was conducted for statistical analyses (d). Source data are provided as a Source Data file. NTC, no template control.
