G box chemical xl system

Each frozen tissue segment was extracted for total protein content using a Precellys 24 bead‐based homogenizer with CK‐28R tubes (Stretton Scientific) as described previously.¹⁵ Protein extracts were aliquoted to flash freeze in liquid nitrogen for PKA activity assays or snap frozen on dry ice for protein quantification using Bradford assay reagent (following manufacturer's protocol; BSA standards used) and immunoblotting. PepTag cAMP‐dependent protein kinase assay kit (Promega) was used to measure basal PKA activity as described previously,¹⁵ when reliability of the assay for myometrial tissues in response to five different cAMP/PKA‐enhancing agents was demonstrated. Agarose gels were imaged using a G:BOX Chemical XL system (Syngene) and analyzed by histogram‐based densitometry using ImageJ v1.5 (National Institutes of Health; Bethesda, MD, USA; Research Resource Identifier (RRID) SCR_003070). Each set of assays included positive (purified PKA catalytic subunit) and background (assay buffer) controls.

Total protein extracts (same as those prepared for PKA activity assays) were used as previously described for detecting Ser16‐phosphorylated and total heat shock protein 20 (HSP20),¹⁵ as well as PR, CAPs (cyclooxygenase‐2 (COX‐2), connexin‐43 (Cx43) and oxytocin receptor (OTR)) and housekeeping proteins (glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) and β‐tubulin)³¹; 10 and 25 μg total protein per sample was used to detect CAPs and PR, respectively. Specificity validation for primary antibodies shown in Figure S2. Western blots were cut to allow simultaneous detection of targets and housekeeping proteins. Chemiluminescence imaging undertaken using a G:BOX Chemical XL system (Syngene). ImageJ v1.5 used for histogram‐based densitometry as described previously.³¹