Westernbreeze kit

Following 6 days of RNAi treatment, cells were scraped and 500μL was collected for immuno-blotting. To extract protein, cells were first pelleted at 5,000xg for 3 minutes at 4°C. Cell pellets were next washed in 100μL 1X phosphate buffered saline before a second centrifugation. The supernatant was removed and the cell pellets were resuspended in 40μL of lysis buffer (50mM Tris-HCl pH 6.8, 150mM NaCl, 0.5% SDS, and 0.5X protease inhibitors (Roche)). After a 5-minute incubation at room temperature, the lysates were vortexed briefly. The samples were then cleared by centrifugation at room temperature at 14,000xg for 10 minutes. The supernatant was transferred to a new tube and the protein abundance was quantified using a Qubit Fluorometer (ThermoFisher Scientific).
To immuno-blot, a total of 5μg of protein was loaded on a pre-cast Tris Glycine gel (ThermoFisher Scientific) and immobilized on PVDF membrane using the iBlot transfer system (ThermoFisher Scientific). CLAMP (1:1000, rabbit, SDIX) and Actin (1:400,000, mouse, Millipore) proteins were detected using the Western Breeze kit (ThermoFisher Scientific) following the manufacturer’s instructions.

Salivary glands from third instar larvae were dissected in cold PBS and samples frozen in liquid nitrogen. Total protein from the samples was extracted by homogenizing tissue in the lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% SDS, 0.5× protease inhibitor) using a small pestle. After a 5 min incubation at room temperature, cleared the samples by centrifuging at room temperature for 10 min at 14,000×g. To blot for CLAMP and Actin, 5 µg of total protein was run on a Novex 10% Tris-Glycine precast gel (Life Technologies). To measure Sex-lethal protein levels, 20 µg of total protein was run on a Novex 12% Tris-Glycine precast gel (Life Technologies). Protein was transferred to PVDF membranes using the iBlot transfer system (Thermo Fisher Scientific) and probed the membranes for CLAMP (1:1000, SDIX), Tubulin (1:5000, Abcam), and SXL (1:500, a gift from Fatima Gebauer) antibodies using the Western Breeze kit following the manufacturer’s protocol (Thermo Fisher Scientific). We quantified the relative expression of protein for SXL using the gel analysis tool in ImageJ software following the website’s guidelines (Schneider et al., 2012 (link)). For each genotype, we first internally normalized the amount of SXL protein to Actin. Next, we determined the protein’s relative expression by comparing the Tubulin normalized quantities to y[1], w[1118] female samples.

After treatment of S2 cells with either clamp or gfp RNAi, we collected a total of 2mL of cells for total RNA (1mL cells) and protein extraction (1mL cells). The preparation of mRNA for qPCR analysis was performed as previously described [7 (link),11 ], with the exception that gapdh was used for internal normalization. Primers used to target amplification have been published previously [7 (link),31 (link)]. The average ΔCt values for clamp or nelf-b transcript was calculated from four biological replicates and significant differences between means were calculated using a T-test.
Total protein was extracted to determine NELF-B abundance after clamp RNAi following the protocol described previously [7 (link),11 ]. Immobilized proteins were blotted for NELF-B (rabbit, 1:1000, gift from Karen Adelman) and detected using the Western Breeze kit (ThermoFisher Scientific). A similar protocol was followed to detect associations between CLAMP and NELF-B after immunoprecipitation. For the detection of NELF-A, proteins were transferred to PVDF membrane using the Xcell II^TM blot module. The Western Breeze kit was then used to detect NELF-A (rabbit, 1:1000, gift from David Gilmour).