Ge amersham imager 600

Western blot assay was conducted as previously described^{18 (link)}. Briefly, cells were rinsed with PBS and resuspended in Western and IP Lysis Buffer (P0013, Beyotime) with 1 mM PMSF. The cell supernatants were collected after centrifugation at 12,000 rpm for 5 min. Protein samples were quantified using the BCA Protein Assay Kit (P0012, Beyotime). A total of 40–80 µg proteins were separated using 8–15% gradient SDS-PAGE gels and transferred onto PVDF membranes (Merck Millipore, Burlington, MA, USA). The membranes were then incubated with 5% fat-free milk, primary antibodies (1:1000), and appropriate HRP-conjugated secondary antibodies (1:5000). Protein expression was visualized using an enhanced ECL detection kit (ORT2655, PerkinElmer, Waltham, MA, USA) and GE Amersham Imager 600 (GE, Chicago, IL, USA). The gray values of the bands were analyzed using ImageJ 1.52a.

Western blot assay was conducted as previously described [23 (link)]. In brief, the tissue samples and cells were homogenized and lysed in RIPA Lysis Buffer (P0013B, Beyotime) and Western and IP Lysis Buffer (P0013, Beyotime) with 1 mM phenylmethylsulfonyl fluoride (PMSF). The cell lysate was centrifuged at 4 °C for 15 min, and the cell supernatant was collected. The concentration of extracted proteins was measured using the bicinchoninic acid (BCA) Protein Assay Kit (P0012, Beyotime). A total of 40–80 µg proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to 0.22 µm polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Burlington, MA, USA). The membranes were incubated with 5% fat-free milk for 1 h at room temperature (RT), followed by incubation with the primary antibodies (1:1000) overnight at 4 °C and appropriate horseradish peroxidase-linked secondary antibodies (1:5000) for 1 h at RT. Immunoreactive species were visualized using enhanced chemiluminescence (ECL) detection Kit (ORT2655, PerkinElmer, Waltham, MA, USA) and GE Amersham Imager 600 (GE, Chicago, IL, USA). β-actin was used as a loading control to normalize protein loading. The intensities of immunoreactive bands were analyzed using ImageJ 1.52a software from the National Institutes of Health (Bethesda, MD, USA).

Standard western blot techniques were used [61 ]. Mcm2-7 was probed with polyclonal UM174 (gift from Bell lab, MIT—[62 (link)]) and anti-rabbit HRP conjugate secondary (Promega W401B), tubulin was probed with monoclonal anti-tubulin antibody (Sigma T5168) and anti-mouse HRP conjugate secondary (Promega W402B), and GFP was probed with the monoclonal JL8 antibody (Takara) and anti-mouse HRP conjugate secondary (Promega W401B). Probing of Mcm2-7 using UM174 antibody was performed using 12% gels in order to compress signal from the six MCMs into a compact region to facilitate quantification. Signal was detected using SuperSignal West Dura chemiluminescent HRP substrate (Thermo 34076) and GE Amersham Imager 600. Quantitation of gel images was carried out using ImageJ (NIH, Maryland, USA).

The proteins in PTC cells were extracted using RIPA lysis buffer (P0013, Beyotime, China) containing protease inhibitor cocktails (FD1001, Fudebio, Hangzhou, China) on ice. Equal amounts of protein lysates were separated by SDS-PAGE gels at 120 V for 1.5 h and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Amersham Bioscience, Piscataway, NJ) at 280 mA for 1.5 h. Membranes were blocked for 1 h with 5% skim milk powder in TBST in tris-buffered saline containing 0.1% Tween 20 and incubated overnight at 4 °C with specific primary antibodies. Then, the membranes were washed with TBST and incubated with an HRP-conjugated secondary antibody (FDM007 and FDR007, Fudebio, Hangzhou, China). The protein bands were visualized by chemiluminescence using a GE Amersham Imager 600 (GE, USA). Anti-beta actin and anti-DDHD2 antibodies were purchased from Proteintech (Chicago, USA). Primary antibody dilution buffer was purchased from Dalian Meilun Biotechnology Co., Ltd. (MB9881, Dalian, China).