Easy 2 protein quantitative kit

Cells were lysed using RIPA buffer (1×) supplemented with cocktail protein inhibitors for 30 min. The concentrations of proteins were determined using Easy II Protein Quantitative Kit (TransGen Biotech, Beijing, China), respectively. Equal amounts of proteins were separated by 8-12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore). Membranes were blocked in non-fat milk and probed with primary antibodies (1:1000) overnight at 4 °C. Then, the membranes were washed and incubated with the appropriate horseradish-peroxidase-coupled secondary antibody for 1 h at room temperature. Finally, protein bands were detected with a SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific) and acquired by Tanon 5200 Imaging System (Tanon, Shanghai, Beijing).

The total protein was obtained from testicular tissue using a ProteinExt^® Mammal Total Protein Extraction Kit (DE101, TransGen Biotech, Beijing, China). An Easy II Protein Quantitative Kit was used to determine the protein concentration (DQ111, TransGen Biotech, Beijing, China). The proteins were separated via SDS separation using polyacrylamide gel electrophoresis and transferred to PVDF membranes (Solarbio, Beijing, China). The membranes were cultured overnight at 37 °C and then with antibodies below 4 °C.
The membranes were washed with TBST (1 L of TBST contains 50 mL of TrisHCL (1 M, pH 7.5); 8 g of NaCl; 0.2 of KCL; 0.5 mL of Twain) and incubated with a secondary antibody blocking solution for 2 h at room temperature [40 (link)]. Proteins were detected using a DNR Bio Imaging system using the NcmECL Ultra method according to the manufacturer’s instructions (Ncmbio, Suzhou, China). The expression of target proteins was quantified using a gel quantification system.

The catalytic domain of PP5 (167-489 amino acids, PP5c) was ligated to the pMD-19T vector. The PaPP5c-pMD-19T plasmid was digested at the Nde1 and Xho1 site and then inserted into pET-30a (+) (TaKaRa) vector (digested at the same restriction sites) with T4 ligase (ThermoFisher). The recombinant plasmid was transformed into E. coli BL21 (DE3) cells. The positive transformant was cultured in LB liquid medium including 50 mg/mL of kanamycin at 37 °C with shaking at 200 rpm until the OD600 reached 0.5. We then added 0.1 mM of IPTG and 1mM of MnCl₂ to induce protein expression at 22 °C, with shaking at 200 rpm for 24 h. Recombinant cells were centrifuged at 4 °C, 8000 g, for collecting the cell pellet. The harvested cell pellet was resuspended in PBS (10 mM), lysed with lysozyme (1 mg/mL), and then disrupted with sonication on ice for 10 min. After sonication, the supernatant was subjected to protein purification. The supernatant was purified with a Ni-NTA column (Smart-Lifesciences, Changzhou, China) using gradient concentrations of imidazole buffer (50–250 mM) to dissolve the protein. The purified protein was detected by 15% SDS-PAGE. Dialysate (4 mM of MnCl₂, 10 mM of Tris-HCl, pH 8.0) was employed to dialyze the protein overnight at 4 °C. The concentration of purified protein was determined by the BCA method using the Easy II Protein Quantitative Kit (TransGene, Beijing, China).

Cells were scraped from plates and lysed in RIPA buffer [50 mM Tris-Cl (PH7.4), 150 mM NaCl, 1 mM EDTA (PH8.0), 0.25% DOC (deoxycholic acid), 10% glycerol, 1% Nonidet P40 and 1% Triton-X100] supplemented with protease inhibitors (Bimake) and phosphatase inhibitors (Bimake). The cell lysates were clarified by centrifugation at 13,000 rpm, for 15 minutes at 4 °C. The protein samples from tumors were homogenized with glass beads and then lysed in RIPA buffer with protease/phosphatase inhibitors. The protein concentrations of the lysates were measured using EASY II Protein Quantitative Kit (Transgene). SDS-PAGE was performed for equal amounts of protein per sample, followed by transferring to a PVDF membrane (Millipore). Then the proteins on the PVDF membrane were immunoblotted with indicated antibodies.

The pMAL-C5X-DmAMP1W and pMAL-C5X were transformed in E. coli DE3 competent cells. The transformed competent cells were grown in 200 mL LB medium (100 μg/mL ampicillin) until OD600 was 0.6, and then transformed competent cells were induced with 0.1 mM concentrations of IPTG under 28 °C for 12 h at 180 rpm.
The culture was divided into 50 mL tubes and then centrifuged at 4000 × g for 20 min to harvest cells. The cells were suspended in PBS buffer (pH 7.4) and divided in 1.5 mL tubes with 10 μL lysozyme (Thermo Fisher Scientific, Boston, MA, USA). Then, the cells were broken up by freeze-thaw method. The cell debris was centrifuged for 15 min at 4 °C and 12,000 × rpm. The clear supernatant, containing soluble fraction, was collected and purified; 100 μL Amylose Resin (NEB, Ipswich, MA, USA) was flowed with PBS buffer (pH 7.4) 3 times, and then the supernatant solution was incubated with Amylose Resin at 4 °C with end-over-end rotation overnight. The MBP and MBP-DmAMP1W were eluted by the MBP elution buffer (0.04% maltose solution), respectively. BCA method was used to confirm the protein concentration referring to the Easy II Protein Quantitative Kit (TransGen Biotech, Beijing, China) instruction. After that, a total of 10 μL of the purified proteins were analyzed by 12% SDS–PAGE (Bio-Rad, Hercules, CA, USA).

After washing with PBS, the transfected cells were treated with a whole cell lysis assay kit (KeyGEN BioTECH, China). Protein concentrations were assessed using a Easy II Protein Quantitative Kit (Transgen, China). Equal quantities of protein were separated on 10% SDS-PAGE and then transmitted to PVDF membranes (Millipore, USA). Following blocking with 5% non-fat milk, the blots were incubated with the relevant antibodies overnight at 4 °C. Finally, the transfer membranes were incubated with secondary antibodies and images of the bands were captured using an ODYSSEY infrared imaging system.

For the preparation of immunoblotting (IB) samples, the protein of cell lysates was quantified using Easy II Protein Quantitative Kit (based on BCA; TransGen Biotech, Beijing, China). Next, the equal quantification of protein samples was electrophoresed using SDS/PAGE gel (containing 1% SDS, 1.5 m Tris pH8.8) and was transferred to nitrocellulose (NC) membrane. After blocking with 5% (w/v) skim milk at room temperature for 1 h, the membranes were incubated with primary antibodies in recommended dilution at 4 °C overnight. The next day, after washing with 1×TBST three times (each for 5 min), the membranes were incubated with HRP‐conjugated secondary antibodies (with a dilution of ~ 1 : 2000–5000) at room temperature for 1 h. After washing with 1×TBST for 3 × 5 min, the membranes were detected using Tanon™ High‐sig ECL Western Blotting Substrate Kit (Cat: 180‐501, Tanon, Shanghai, China). The protein bands were scanned and analyzed using Bio‐Rad ChemiDoc MP. Primary antibodies of HNRNPA0 (Cat: #5545), LIN28A (Cat: #8706), FLAG (Cat: #14793), COL5A1 (Cat: #86903), MMP1 (Cat: #54376), and GAPDH (Cat: #5174) were purchased from Cell Signaling Technology (Danvers, MA, USA). SPINK5 (Cat: AF8515) was purchased from R&D System (Minneapolis, MN, USA).