Ecl western blotting protocol

Western blots were performed from the cells transfected with miR-3648 overexpressing plasmids or ant-3648 after 48 h. While Western blots for TG treatment cells were performed at the indicated times post treatments, wor Western blots, samples were separated on 10% SDS–PAGE gels and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). Proteins on the membranes were blocked with 5% non-fat milk for 1 h at room temperature. The membranes were then incubated with primary antibodies in 5% non-fat milk over night at 4 °C. After washing the membranes three times with TBST, these were incubated with the respective secondary antibodies. Membranes were processed according to the ECL Western blotting protocol (GE Healthcare). The following primary antibodies were used in Western blots: anti-APC2 (Abcam, cat#113370) and anti-Actin (Signalway antibody, 21338).

For preparation of samples, protein extracted from testicular tissues was minced and lysed in RIPA buffer (Beyotime Biotech, Shanghai, China) on ice more than 20 mins. After sonication, the concentrations were measured with a BCA protein assay kit (Beyotime Biotech). Total protein was loaded onto 15% SDS-PAGE gels and then transferred to PVDF membranes. Membranes were processed according to the ECL Western blotting protocol (GE Healthcare, General Electric Company, Boston, USA). Quantification of the band intensities was performed with ImageJ (NIH, Bethesda, MD, USA). An area above each band, the same size as the corresponding band, was used for background subtraction. The following antibodies were used in Western blots: anti-Nanos2 (1:200; GeneTex, Irvine, CA, USA) and anti-GAPDH (1:1000; TransGen Biotech, Beijing, China).

After the different treatments, cells were washed twice with PBS 1x and lysed for protein extraction according to standard procedures. Alternatively, for determining the effect of the extraction method on the capacity to detect prelamin A in Zmpste24 KO cells, we proceed to sonication before protein concentration determination. Protein concentration was achieved by the Bradford method, using de Bio-Rad^® (Hercules, CA, USA) reagent and BSA as standard. Equal amounts of protein (15–20 μg) were submitted to electrophoresis and after SDS-PAGE gels were transferred to Immobilon P PVDF membranes (Merck Millipore, Burlington, MA, USA). Then, membranes were blocked with 5% BSA and incubated overnight with primary antibodies at 4ºC. The corresponding bands were visualized using the ECL Western blotting protocol (GE Healthcare, Little Chalfont, UK). Alternatively, for studying the role of cell cycle in prelamin A elimination, cells were cultured under normal growing conditions until reaching 70–80% of confluency. After that, the medium was discarded and we added a new medium with all the components apart from serum, which was added at 0,1% for 24 h. Then, cells were lysed, and the protocol was the same as previously described.

For western blotting, samples were separated by 10% SDS-PAGE and electrophoretically transferred to PVDF membranes (Millipore). The membranes were processed according to the ECL western blotting protocol (GE Healthcare) and scanned with Amersham Imager 680 (GE Healthcare). The following primary antibodies were used: anti-EGFR (BBI, D260292); anti-PI3K (BBI, D155308); anti-AKT (Proteintech, 10,176–2-AP); anti-p-PI3K Tyr458 (Cell Signaling Technology, 4228S); anti-p-AKT Ser473 (Proteintech, 66,444–1-Ig); anti-c-MYC (Proteintech, 10,828–1-AP) and anti-β-actin (BBI, D110001). The anti-β-actin antibody was used as an endogenous control for normalization. Antibody validation is provided on the manufacturers’ websites.

After the different treatments, cells were washed twice with PBS and lysed for protein extraction according to standard procedures. Protein concentration determination was achieved by the Bradford dye method, using the Bio-Rad (Hercules, CA) reagent and BSA as standard. Equal amounts of protein (15–20 µg) were submitted to electrophoresis and after SDS–PAGE gels were transferred to Immobilon P PVDF membranes (Merck–Millipore). Then, membranes were blocked with 5% non-fat dried milk and incubated overnight with antibodies at 4 °C. The corresponding bands were visualized using the ECL Western blotting protocol (GE Healthcare, Little Chalfont, UK). All the blots presented in the paper are non-saturated signals being at the linear range of exposition with the following exceptions: in the blots from the Fig. 3a we present a long exposure to compare the differential effect of either metformin or glucose deprivation on phospho-p70S6K (Thr 389) and p70S6K in MIN6 and MEF cells. Similarly, in Fig. 4a–c, we show a long exposure of phospho-P70S6K (Thr 389) or phospho-S6 (Ser 235/236) to see the hyperactivation of MTORC1 signaling and the inhibitory effect of the energetic stressors on MTORC1 signaling.