Ab31307

The antibodies were used as follows: mouse monoclonal antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, MAB374, Merck, 1:10000); rabbit polyclonal phosphorylated form of cAMP-dependent protein kinase (pPKA (Thr197), 44988A, Thermo Fisher Scientific, 1:1000); rabbit polyclonal to the recombinant fragment corresponding to a region within amino acids 1 and 351 of PKA catalytic subunit alpha (PA5-21842, Invitrogen, 1:1000); mouse monoclonal to phosphorylated form of cAMP response element-binding protein (CREB (Ser 133), 9196S, Cell Signaling, 1:500); rabbit monoclonal to cAMP response element-binding protein (CREB, 9197S, Cell Signaling, 1:1000); rabbit polyclonal to adenylate cyclase 3 (AC3, PA1-31191, Invitrogen, 1:250); rabbit polyclonal to adenylate cyclase 7 (AC7, PA5-103390, Thermo Fisher Scientific, 1:500); mouse monoclonal to A-kinase anchoring protein 9 (AKAP9, ab32679, Abcam, 1:125); goat monoclonal to A-kinase anchoring protein 9 (AKAP9, ab31307, Abcam, 1:200); rabbit monoclonal to cAMP-specific 3′,5′-cyclic phosphodiesterase 4D (PDE4D, ab171750, Abcam, 1:1000); HRP rabbit polyclonal to β-tubulin (ab21058, Abcam, 1:10000); rabbit polyclonal to MVI (25–6791, Proteus, 1:500); goat anti-mouse IgG antibody, HRP conjugate (AP308P, Millipore, 1:10000); goat anti-rabbit IgG antibody, HRP conjugate (AP307P, Millipore, 1:10000).

Hindlimb muscle cross-sections of newborn animals were air-dried for 30 min and were rinsed with 1X PBS for another 15 min. After rinsing, the sections were put in 4% PFA for 30 min. After three wash steps with 1X PBS for 5 min, the sections were incubated with NH4Cl for 30 min followed by blocking in 1X PBS with 0.3% Triton X-100 and 5% horse serum (26050088, Gibco) at 4°C for 1 h. Next, the sections were incubated overnight with antibodies against AKAP9 (ab 31307, Abcam, 1:50) prepared in a blocking solution at 1:125 dilution. Following incubation with the primary antibodies and after rinsing 3 times with 1X PBS with 0.3% Triton X-100, the sections were incubated in blocking solution at room temperature with Alexa Fluor 555-conjugated secondary antibodies to detect primary antibodies (A21432, Invitrogen, 1:1000). Finally, the sections were washed 3 times in 1X PBS with 0.3% Triton X-100 and mounted using Vectashield Plus antifade mounting medium with DAPI (H-2000, Vector Laboratories).