We dissected salivary glands from third instar larvae in cold PBS and froze samples in liquid nitrogen. We extracted total protein from the samples by homogenizing in lysis buffer (50mM Tris-HCl pH 8.0, 150mM NaCl, 1% SDS, 0.5X protease inhibitor) using a small pestle. After a five-minute incubation at room temperature, we cleared the samples by centrifuging at room temperature for 10 minutes at 14,000 × g. To blot for CLAMP and Actin, we ran 5 micrograms of total protein on a Novex 10% Tris-Glycine precast gel (Life technologies). We transferred proteins to PVDF membranes using the iBlot transfer system (ThermoFisher Scientific) and probed the membranes for CLAMP (1:1000, SDIX) and Actin (1:400,000, Millipore) using the Western Breeze kit following the manufacturer’s protocol (ThermoFisher Scientific).
Relative expression of protein for CLAMP was quantified using the gel analysis tool in ImageJ software following the guidelines outlined on the website (Schneider et al., 2012 (link)). For each genotype, we first internally normalized the amount of CLAMP protein to Actin. Next, we determined relative expression of protein by comparing the Actin normalized quantities to sex of respective y1, w1118;+; (y−w−) sample.
Relative expression of protein for CLAMP was quantified using the gel analysis tool in ImageJ software following the guidelines outlined on the website (Schneider et al., 2012 (link)). For each genotype, we first internally normalized the amount of CLAMP protein to Actin. Next, we determined relative expression of protein by comparing the Actin normalized quantities to sex of respective y1, w1118;+; (y−w−) sample.