Supersignal west femto maximun sensitivity substrate

The procedures were performed as described previously [18 (link)]. Briefly, total protein of cells and fresh tissues were isolated and separated by 6%–8% SDS-PAGE gels. Following the standard procedure, the blocking proteins in the nitrocellulose filter membranes were incubated with primary antibodies against the following antigens: XBP1 (Santa Cruz Biotechnology, Dallas, TX, USA), HIF1α (Santa Cruz Biotechnology), PIK3R3 (Abgent, San Diego, CA, USA), mTOR (Abgent), β-Actin (Sigma-Aldrich, New York, NY, USA). The secondary antibody was horseradish peroxidase-conjugated anti-rabbit IgG (Sigma-Aldrich). SuperSignal West Femto Maximun Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA, USA) was used in the subsequent visualization.

Cleared cell lysates were boiled for 5 min at 95°C and loaded on 8–12% SDS polyacrylamide gels. As a protein marker the Precision Plus Protein All Blue Prestained Protein Standards (1610373, Biorad) was used. After SDS PAGE, the proteins were transferred on a nitrocellulose membrane (Premium 0.45 μm, 10600003, Amershan) for 70 min at 350 mA in transfer buffer. Depending on the antibody, unspecific binding was blocked by incubating the membrane in either 5% milk in PBS-T or TBS-T buffer or in 5% bovine serum albumin (BSA) in TBS-T buffer. The membranes were incubated with the primary antibody on a roller at 4°C over night or 1 h at RT. After three washing steps in the corresponding buffer, the membranes were incubated for 1 h at RT in the secondary horseradish peroxidase (HRP)-conjugated antibody. Primary and secondary antibodies used for western blot are listed above (section antibodies). To visualize the specific detection of proteins, the membranes were developed in a LAS-3000 Imager (Fujifilm) using either self-made enhanced chemiluminescence (ECL) solution 1 and 2, which were mixed in a ratio of 1:1, or the SuperSignal West Femto Maximun Sensitivity Substrate (34096, Thermo Scientific).

Cellular proteins were extracted from cultured cells with a mixture of T-PER Protein Extraction Reagent (Thermo), PhosSTOP (Roche) and Complete Mini (Roche). Protein samples were separated in sodium dodecyl sulfate (SDS)-PAGE and transferred to nitrocellulose filter membranes (Millipore, USA). After blocking in phosphate buffered saline (PBS) / Tween-20 containing 5% nonfat milk, the membranes were incubated with the following primary antibodies: AKT (Cell Signaling Technology), PTEN (Cell Signaling Technology), ERK1/2 (Cell Signaling Technology), phosphor-AKT (ser473)(Cell Signaling Technology), phosphor-ERK1/2(thr202/tyr204)(Cell Signaling Technology). β-Actin(Sigma-Aldrich). HRP-conjugated anti-rabbit IgG (Sigma-Aldrich) were incubated as the secondary antibodies. Subsequent visualization was detected with Super Signal West Femto Maximun Sensitivity Substrate (Thermo, Japan).

Aliquots of 1−2 x 10⁶ WT or sAC^−/− double transgenic sperm were incubated for 60 min in TYH buffer in the absence or presence of 5 mg/mL BSA and 20 mM NaHCO₃ or 0.5 mM 8Br-cAMP and 0.5 μM IBMX. Sperm were collected by centrifugation for 3 min at 2000 x g, washed twice with 1 mL TBS, and resuspended with Laemmli sample buffer without β-mercaptoethanol, vortexed for 10 s and boiled for 3 min. After centrifugation, 5% β-mercaptoethanol was added to the supernatants and boiled again for 5 min. Protein extracts equivalent to 0.5−1 x 10⁶ sperm/lane were subjected to SDS-PAGE and electrotransferred to PVDF membranes at 250 mA for 90 min. Membranes were blocked with 3% BSA in TBS containing 0.1% Tween-20 (T-TBS). Antibodies were diluted in T-TBS as follows: 1/5,000 for anti-pTyr and 1/3,000 for anti-pPKA substrates. Secondary antibodies were diluted 1/10,000 in T-TBS and the membranes analyzed using an enhanced chemiluminescence detection kit (SuperSignal West Femto Maximun Sensitivity Substrate, Thermo Scientific) according to manufacturer’s instructions.

After washing in PBS, cells were lysed in SDS lysis buffer (62.5 mM Tris-HCl pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 50 mM DTT, bromophenol blue). Samples were centrifuged for 10 minutes at 17.949 xg and 4°C to pellet cell debris. If necessary, the lysates were sonicated for a few seconds before centrifugation. Protein concentrations were measured using a NanoDrop1000 (Peqlab, VWR, Darmstadt, Germany). The cleared cell lysates were boiled for 5 minutes at 95°C and loaded on 8% to 12% SDS polyacrylamide gels. The Precision Plus Protein All Blue Prestained Protein Standards (1610373, Bio-Rad, Feldkirchen, Germany) was used as a protein marker. After SDS PAGE, the proteins were transferred on nitrocellulose membranes, unspecific binding was blocked by incubating the membranes in 5% milk in PBS-T buffer, and the primary antibody incubation was performed on a roller incubator at 4°C overnight or 1 hour at room temperature (RT). After 3 washing steps in PBS-T, the membranes were incubated for 1 hour at RT with the secondary horseradish peroxidase (HRP)-conjugated antibody. To visualize the detection of specific proteins, the membranes were developed in a LAS-3000 Imager (Fujifilm, Duesseldorf, Germany) using either the SuperSignal West Femto Maximun Sensitivity Substrate (34096, Thermo Fisher Scientific, Waltham, USA) or self-made enhanced chemiluminescence (ECL).