Stain free tgx gradient gels

Cells were lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris- HCl, pH 8.0) supplemented with protease Inhibitor Complete Mini (Roche, 000000011836170001) on ice for 15 min. The cell lysates were rotated at 4 °C for 30 min and the insoluble material was removed by centrifugation at 15,000 g for 15 min. Protein concentrations were determined by Bio-Rad Protein Assay using bovine serum albumin (BSA) as the standard. Following the normalization of protein concentrations, lysates were mixed with an equal volume of 2X Laemmli sample buffer and incubated for 5 min at 95 °C prior to separation by SDS PAGE on stain-free TGX gradient gels (Bio-Rad). Following SDS-PAGE, the proteins were transferred to polyvinylidene fluoride membranes (300 mA for 90 min at 4 °C). The membranes were then blocked with BSA (Sigma-Aldrich) dissolved in PBS/Tween-20 (3% BSA, 0.5% Tween-20 for 1–2 hours), followed by immunoblotting with the primary antibody specified for each experiment for PIWIL2 (Abcam, ab181340 diluted at 1:1000), PIWIL4 (Abcam ab 111714, diluted at 1:1000), and beta Actin (Abcam ab1801, diluted at 1:1000). After the washing steps, the membranes were incubated with goat anti-rabbit IgG (H + L) HRP-conjugated secondary antibodies (Bio-Rad) and detected using ECL (Amresco). Densitometry was performed using Image Lab software v. 4.1 (Bio-Rad).

Cells were lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris- HCl, pH 8.0) supplemented with protease Inhibitor Complete Mini (Roche) on ice for 15 min. The cell lysates were rotated at 4°C for 30 min and the insoluble material was removed by centrifugation at 14,000 rpm for 15 min. Protein concentrations were determined by BioRad Protein Assay (BioRad) using bovine serum albumin (BSA) as a standard. Following the normalization of protein concentrations, lysates were mixed with an equal volume of 2X Laemmli sample buffer and incubated for 5 min at 95°C prior to separation by SDS PAGE on stain free TGX gradient gels (BioRad). Following SDS PAGE, the proteins were transferred to polyvinylidene fluoride membranes (300 mA for 90 min at 4°C). The membranes were then blocked with BCA (SIGMA) proteins dissolved in PBS/Tween-20 (3% BCA, 0.5% Tween-20 for 1–2 h), followed by immunoblotting with the primary antibody specified for each experiment (Bax abcam ab32503; BCL2 abcam ab59348, beta actin abcam ab1801, and caspase 8 santa cruz sc-81661). After the washing steps, the membranes were incubated with goat anti-rabbit IgG (H+L) or with goat anti-mouse IgG (H+L) HRP-conjugated secondary antibodies (BioRad) and detected using ECL (Pierce). Densitometry was performed using Image Lab software v. 4.1 (BioRad).

Cells were lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris- HCl, pH 8.0) supplemented with protease Inhibitor Complete Mini (Roche) on ice for 15 min. The cell lysates were rotated at 4 °C for 30 min and the insoluble material was removed by centrifugation at 15,000 g for 15 min. Protein concentrations were determined by BioRad™ Protein Assay using bovine serum albumin (BSA) as a standard. Following the normalization of protein concentrations, lysates were mixed with an equal volume of 2X Laemmli sample buffer and incubated for 5 min at 95 °C prior to separation by SDS PAGE on stain-free TGX gradient gels (BioRad). Following SDS-PAGE, the proteins were transferred to polyvinylidene fluoride membranes (300 mA for 90 min at 4 °C). The membranes were then blocked with BSA (Sigma-Aldrich) dissolved in PBS/Tween-20 (3% BSA, 0.5% Tween-20 for 1–2 h), followed by immunoblotting with the primary antibody specified for each experiment CFTR (Merck MM13–4 diluted at 1:1000), HIF-1α (Abcam ab16066, diluted at 1:1000); and beta ACTIN (Abcam ab1801, diluted at 1:1000). After the washing steps, the membranes were incubated with goat anti-rabbit IgG (H + L chains) or with goat anti-mouse IgG (H + L) HRP-conjugated secondary antibodies (BioRad) and detected using ECL (Amresco). Densitometry was performed using Image Lab software v. 4.1 (BioRad).