Ab18197

The protein level of TRIM16 was detected using Western blot, according to the experimental procedures recorded in the previous publications [29 (link)]. Briefly, transfected cells were lysed in Radioimmunoprecipitation assay (RIPA) lysis buffer with protease inhibitor (Thermo Fisher Scientific) to extract total protein. The protein quantification was measured by the BCA^TM Protein Assay Kit (Pierce, Appleton, WI, USA). Then the equivalent protein (50 µg) was added to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to obtain the target protein. Next, the target proteins in the gel were transferred onto polyvinylidene difluoride (PVDF) membranes and incubated with primary antibodies against TRIM16 (ab72129, 1:2000, Abcam, Cambridge, MA, USA), hexokinase 2 (HK2, ab227198, 1:5000, Abcam), B-cell lymphoma protein 2 (Bcl-2)-associated X (BAX, ab32503, 1:2000, Abcam), Matrix metalloproteinase 2 (MMP2, ab92536, 1:1000, Abcam), Proliferating cell nuclear antigen (PCNA, ab18197, 1:1000, Abcam), and β-actin at 4°C overnight. The membranes were then incubated with second antibodies (Horseradish peroxidase-conjugated IgG antibody). Finally, the Western blot intensities were detected using LAS 4000 Image Reader (Fujifilm, Tokyo, Japan)

Protein expressions were determined using Western blot assay [33 (link)]. Total protein was extracted using RIPA cell lysate buffer containing a protease inhibitor cocktail. The protein concentration was then measured using the BCA method. The proteins were separated by SDS-PAGE gel electrophoresis and transferred to PVDF membranes. The membranes were then blocked with 5% BSA at room temperature for 50 min to eliminate nonspecific binding of primary and secondary antibodies. Primary antibody (cleaved-Caspase3 antibody, ab32042, 1:1000, Abcam; GAPDH antibody, ab9485, 1:1000, Abcam; PCNA antibody, ab18197, 1:1000, Abcam; CCND2 antibody, ab207604, 1:1000, Abcam;) was then added to the PVDF membrane and incubated overnight at 4°C. The next day, the membranes were probed with the corresponding HRP-labeled secondary antibody (ab7090, 1:1000, Abcam) by incubation at room temperature for 2 h. ECL chromogenic solution A and B were mixed, added dropwise to the position of the target band on the membrane, and photographed using ImageJ software.