Ab190479

The physical association of SIRT3 and LCAD was detected by immunoprecipitation analysis. In brief, whole cell extracts were prepared by lysing cells in lysis buffer (50 mM Tris-HCl pH 7.4, 180 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, and 1X Halt Protease and Phosphatase Inhibitor Cocktail). Immunoprecipitation experiments were carried out using the antibody specific to LCAD (17526-1-AP; Proteintech) and Ezview Red Protein G Affinity Gel (Sigma-Aldrich Corp.). Typically, 500 μg of lysate was incubated overnight with 1 μg of the abovementioned primary rabbit polyclonal antibody to LCAD at 4 °C. Immunoblots were then probed with LCAD antibody.
SIRT3 (1:1000; D22A3; Cell Signaling Technology) and acetyl-lysine (1:1000; ab190479; Abcam) expression were determined in the same membrane after stripping off the immune complex for the detection of LCAD. In parallel, immunoprecipitation assays using rabbit monoclonal antibodies reactive to IgG were used as controls. The results of SIRT3 and acetyl-lysine after LCAD immunoprecipitation were normalized with those obtained using rabbit monoclonal antibodies reactive to IgG immunoprecipitation assays as well as with the LCAD total levels. The initial input was also used as a loading control of LCAD in immunoprecipitation analysis (Supplementary Materials).

Proteins from cell lysates were incubated with an anti-KAT5 antibody (1:500, ab300521; Abcam, Cambridge, UK), anti-ALDOA antibody (1:300, ab181662; Abcam), or anti-IgG antibody (as an NC; 1:500, ab172730; Abcam) at 4 °C overnight. The immune complexes were incubated with Protein G/A beads under rotation at 4 °C for 3–5 h. The bead-bound complexes were collected by centrifugation at 1000 × g and 4 °C for 5 min and washed thrice with washing buffer (50 mM Tris-HCl [pH 7.4], 100 mM NaCl, 5 mM CaCl₂, 5 mM MgCl₂, and 0.1% Nonidet P-40). Eluted immune complexes were resuspended in 1 × SDS-PAGE loading buffer, denatured in a metal bath at 100 °C for 5 min, and then loaded on 10% polyacrylamide gels for electrophoresis. After separation, the proteins were transferred onto a PVDF membrane and detected using the western blotting method with an anti-ALDOA antibody (1:2000, ab181662; Abcam) and anti-acetyl Lysine antibody (1:1000, ab190479; Abcam).

The concentration of total protein from lysed cells was determined with a BCA kit (23227; Thermo Fisher Scientific). Protein samples were diluted in 5 × sample buffer, electrophoresed on 12% separating gels for 1.5 h, and transferred onto a membrane, which was then immersed in PBS blocking solution containing 5% (w/v) non-fat powdered milk for 60 min at ambient temperature. After that, the membrane was incubated overnight at 4 °C with anti-ZNF692 (1:1500, ab204595), anti-KAT5 (1:1500, ab300521), anti-ALDOA (1:1500, ab252953), anti-acetyl Lysine (1:1500, ab190479), and anti-GAPDH (1:1500, ab9485) (all from Abcam) antibodies. After washing, the membrane was placed in secondary antibody solution (1:1500, ab150077; Abcam) for 1 h at ambient temperature and imaged using a BioSpectrum system (UVP, USA).

Samples and cells were collected for Western blotting as previously described. Western blot analysis was performed using the following antibodies: LHPP (NBP1-83273, Novus, 0.2 ug/ml), METTL14 (ab220030, Abcam, 1:1000), HIF-1α (ab243861, Abcam, 1:1000), β-ACTIN (ab8226, Abcam, 1:2000), Acetyl Lysine (ab190479, Abcam, 1:1000), P300 (ab10485, Abcam, 1:5000), TIP60 (ab151432, Abcam, 1:1000), GCN5 (ab282176, Abcam, 1:1000), PCAF (ab176316, Abcam, 1:1000), GLUT1 (ab115730, Abcam, 1:5000), c-Myc (ab32072, Abcam, 1:1000), PKM2 (ab137852, Abcam, 1:1000), ALDOLASE (ab252953, Abcam, 1:1000), ENOLASE1 (H00002023-M01, Novus, 1:500), GLS1 (H00002744-M01, Novus, 1:500), GSK-3β (phospho S9) (ab75814, Abcam, 1:5000), GSK-3β (ab32391, Abcam, 1:5000), β-CATENIN (phospho S37) (ab75777, Abcam, 1:500).

Protein lysis buffer (ZD409, ZOMANBIO, Beijing, China) and a Total Protein Extraction Kit (BC3711, Solarbio, Beijing, China) were employed to extract total proteins from HCC cells. The obtained proteins were isolated by SDS–PAGE (P1200, Solarbio) and then transferred onto PVDF membranes (T2234, Thermo Fisher). After blocking with 5% skimmed milk, the membranes were cocultured with primary antibodies, including anti-HIF-1α (ab1, Abcam), anti-β-actin (ab179467, Abcam), anti-MMP2 (ab92536, Abcam), anti-MMP9 (ab76003, Abcam), anti-ZO-1 (ab276131, Abcam), anti-E-cadherin (ab40772, Abcam), anti-N-cadherin (ab76011, Abcam), anti-vimentin (ab92547, Abcam), anti-Ac-K (ab190479, Abcam), anti-FOXA3 (PA1-813, Abcam), anti-HDAC2 (ab32117, Abcam), anti-PKM2 (ab137791, Abcam) and anti-DADACT3 (ab797, Abcam). Subsequently, the membranes were incubated with secondary antibody for 1 h. The protein levels were measured by means of an enhanced chemiluminescence detection system. β-actin served as the internal reference.

Acetylation of sperm proteins in both unilateral (n = 6) and bilateral varicocele patients (n = 6) was demonstrated using WB. Immunoprecipitation of acetylated proteins was carried out using anti-acetyl Lysine antibody (ab190479, Abcam, USA) followed by WB detection of selected acetylated proteins. The criteria applied for the selection of DEPs involved in the acetylation process were as follows: (i) Proteins involved in the networks; (ii) abundance of the protein must be moderate or high in any one group; and (iii) proteins with a well-described function in the literature. Four proteins (ANXA2, HIST1H2BA, SERPINB6, and SOD1) were chosen for validation by WB in both the unilateral and bilateral varicocele group.
Immunoprecipitated acetylated proteins were first loaded into a 4–15% SDS–PAGE for 2 h at 90 V. The resolved proteins were transferred onto polyvinylidene difluoride (PVDF) membranes and analyzed as described earlier [52 (link)]. The expression levels of the WB-validated proteins were normalized against the global acetylated proteins (Supplementary Figure S1) and compared between unilateral and bilateral varicocele using the Mann–Whitney test and p < 0.05 was considered significant. Data analysis was performed using MedCalc Statistical Software (version 17.8; MedCalc Software, Ostend, Belgium).

Brains were homogenized in RIPA buffer (50 mM Tris·HCl, pH 7.5, 500 mM NaCl, 2.5 mM MgCl₂, 1% NP-40, 10% glycerol) containing proteinase inhibitors; 30 μg of protein was separated by SDS/PAGE and transferred to PVDF membrane for immunoblot analyses. The membranes were blocked with 5% nonfat dry milk in PBS containing 0.1% Tween 20 (PBST) and probed with primary antibodies recognizing acetylated lysine (1:2000; rabbit monoclonal; Abcam (ab190479), Cambridge, UK), α-tubulin (1:1000; rabbit polyclonal; Cell Signaling Technology (2144S), Danvers, MA), and HDAC6 (1:100; goat polyclonal; Santa Cruz Biotechnology (sc-5258), Dallas, TX). Membranes were incubated with primary antibody overnight at 4 °C, washed three times with PBST, followed by incubation with horseradish peroxidase-conjugated anti-rabbit (Jackson ImmunoResearch (111–035-144), West Grove, PA), anti-goat (Jackson ImmunoResearch (111–035-144)), or anti-β-actin antibodies (Sigma-Aldrich (A3854)) at room temperature for 2 h. Membranes were then washed three times with PBST. Immunoreactivity for each protein was visualized using a chemiluminescence detection kit (GE Healthcare Life Sciences, Little Chalfont, UK). The images were acquired using ImageQuant LAS 4000 (GE Healthcare Life Sciences) and densitometrically analyzed using ImageJ software.