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. 2021 Mar 28;296:100606. doi: 10.1016/j.jbc.2021.100606

Figure 6.

Figure 6

PDE1C interaction and co-localization with PKA, AKAP79, Orai1, and ADCY8 in HASMC and HASMC LEPs.A, representative immunoblots of anti-PDE1C, or anti-IgG, generated immune complexes analyzed for PDE1C, gravin, AKAP79, PKA-RIIβ, PKA-RIα, Orai1, or STIM1. Proteins isolated along with PDE1C in anti-PDE1C immunoprecipitation experiments, but not in identical anti-IgG immunoprecipitations are indicated (arrows, right side). n = 3 experiments in which distinct HASMC cell lysates were treated identically. B and C, representative high-resolution confocal images of HASMCs, transfected with either myc-Orai1, GFP-STIM1, HA-ADCY8, or FLAG-PDE1C plated on coated coverslips (B) or Fluoroblok membranes (C). Transfected HASMCs plated on coverslips were incubated with primary antibodies for individual expression tags (i.e., myc, GFP, HA, FLAG) or with an anti-AKAP79 antibody. Staining of the expressed proteins, or of endogenous AKAP79, are shown in green while actin tetramethylrhodamine-isothiocyanate-conjugated phalloidin is shown in red. C, HASMCs, transfected with either myc-Orai1, GFP-STIM1, HA-ADCY8, or FLAG-PDE1C were plated on Fluoroblok membranes and allowed to accumulate LEPs on the underside of the membranes. LEP on the underside of Fluoroblok membranes were stained with anti-myc, anti-GFP, anti-HA, anti-FLAG, or anti-AKAP79; visualized by staining with 488-conjugated secondary for anti-myc, anti- GFP, anti-HA, anti-FLAG and anti-AKAP79 and tetramethylrhodamine-isothiocyanate-conjugated phalloidin to visualize F-actin and DAPI for nuclei; scale bars, 20 μm, n = 5 to 7 transfections were analyzed per condition. Arrowheads indicate staining of each protein and their accumulation within LEPs. ADCY, adenylyl cyclase; HASMC, human arterial smooth muscle cell; LEP, leading-edge protrusion; PDE, phosphodiesterase; PDE1C, phosphodiesterase 1C; STIM1, stromal interaction molecule 1.