Hrp conjugated goat anti rabbit igg h l

Cells and tissue homogenates were harvested and lysed in RIPA buffer (50 mM Tris-HCl [pH 7.4], 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 1 mM Na₃VO₄, and 1 mM NaF). The protein concentration in the lysate was quantified using the BCA Kit. The cell lysates were loaded onto an SDS-PAGE gel for electrophoresis. After transfer of the proteins onto a piece of nitrocellulose membrane, targets were detected by western blotting using primary antibodies, including NEK6 antibody (1 : 2000, catalog number: 10378-1-AP, Proteintech, Wuhan, China), GAPDH antibody (1 : 10,000, catalog number: 10494-1-AP, Proteintech), secondary antibody, HRP-conjugated goat anti-mouse IgG (H + L) (1:10,000, catalog number: ab205719, Abcam, United States), HRP-conjugated goat anti-rabbit IgG (H + L) (1:10,000, catalog number: ab205718, Abcam), and an ECL Chemiluminescent Substrate Reagent Kit (Invitrogen, catalog number: WP20005, Shanghai, China). Protein expression levels were quantified using ImageJ software. The experiments were performed in triplicate.

All the proteins were obtained from AML tissues and cells using the RIPA lysis buffer and were quantified with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA). SDS-PAGE (8%) was used to separate the protein, and the concentration of loaded protein samples was 40 μg/lane. After performing electrophoresis at constant pressure, the separated protein bands were electro-transferred onto the PVDF membrane (Millipore, USA) with constant current. The membrane was then washed with PBST (0.1% Tween-20/PBS solution) three times and blocked with 5% BSA for 2 h at room temperature. Subsequently, the rabbit polyclonal anti-DOCK1 (Cat#: ab97325, Abcam, USA) and rabbit monoclonal anti-GAPDH (Cat#: EPR16891, Abcam, USA) were employed for primary antibody incubation overnight at 4 °C. HRP-conjugated Goat anti-rabbit IgG H&L (Cat#: ab181602, Abcam, USA) was used for secondary antibody incubation for 2 h at room temperature. After repeated washing with PBST, the blots were visualized using the enhanced chemiluminescence kit (Thermo Fisher Scientific, USA).

U-251 MG and U-87 MG cells were lysed using RIPA buffer (Vazyme). The protein concentrations of samples were measured using a bicinchoninic acid (BCA) protein quantification kit (Abcam, Shanghai, China). Samples (12 μg/lane) were loaded into a 12% SDS-PAGE gel for electrophoresis and then transferred onto a PVDF membrane (Roche, Basel, Switzerland). The membrane was incubated with primary antibody Anti-USP32 (1:1000, CAT#ab251903, Abcam) or Anti-GAPDH (1:2000, CAT#ab9485, Abcam) at 4 °C overnight, followed by incubation with secondary antibody HRP-conjugated Goat Anti-Rabbit IgG H&L (1:2000, CAT#ab6721, Abcam) at 28 °C for 30 min. The signals were visualized using the ECL detection system (Thermo Fisher Scientific, Waltham, MA, USA) and quantified by densitometry using Image J v1.48u.

Mammalian cells cultured near confluence in T75 flasks were washed twice with cold PBS and scraped into 200 µl of Pierce 1x RIPA buffer (Thermo Scientific), supplemented with protease inhibitor cocktail (Millipore Sigma), and then transferred to 1.5 ml microtube kept on ice. Cells were vortexed briefly and disrupted by trituration 10× with a 23 G ¼ inch syringe needle (Becton, Dickinson and Co. Thermo Scientific). To remove unbroken cells and cellular debris, lysates were spun at 17,000 × g for 10 min and the supernatant was collected for downstream applications, as well as for determination of protein concentration by the BCA Protein Assay Kit (Thermo Scientific).
For running Western blots, 30 µg of cell lysates in Sample Buffer was loaded per each well of 4–20% Precast Protein Gel (BioRad, Hercules, CA) and then transferred to PVDF membrane. For blocking, 3% BSA (Sigma-Aldrich) in TBS buffer was used, while antibodies were diluted in 1% BSA in TBS-T. For detection of CD63 protein, we used either anti-mouse CD63 (ab217345) or anti-human CD63 (ab134045) (Abcam, Waltham, MA); for detection of NHE1, the Anti-Na+/H+ Exchanger 1 (extracellular) was used from Alomone Labs (ANX-010, Jerusalem, Isreal); for secondary antibodies, the HRP-conjugated goat anti-rabbit IgG H&L (ab6721, Abcam) was used; primary antibodies were used at 1:2000 and secondary antibodies at 1:10,000 dilutions.