Image lab software

Western blot was performed using the Invitrogen™ Western Devices Benchtop Bundle (Invitrogen™, Catalog #: IW3000S) as per the manufacturer’s protocol. The primary antibody used for PRMT5 detection was the Invitrogen™ PRMT5 Recombinant Rabbit Monoclonal Antibody (ST51-06) (Invitrogen™, Catalog #: MA5-32160) at a 1:1000 dilution. The secondary antibody used for PRMT5 detection was the Invitrogen™ Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP (Invitrogen™, Catalog #: 31460) at a 1:5000 dilution. Beta-actin at a 1:5000 dilution, as well as the Invitrogen™ No-Stain™ Protein Labeling Reagent (Invitrogen™, Catalog #: A44449), were used for normalization as per the manufacturer’s protocol. A Bio-Rad™ ChemiDoc™ MP Imaging System was used for blot imaging. The band intensities were quantified using Bio-Rad’s™ Image Lab software. Bio-Rad’s™ Image Lab software was also used for normalization by normalizing the quantified intensity of each band to the quantified intensity of its respective housekeeping protein (beta-actin) or total protein. Data analysis was performed by dividing the normalized band intensities of the CRC cell lines by the normalized band intensity of the control normal colon epithelial CCD 841 CoN cell line to arrive at the fold changes.

For experimental verification of SL protein interactions with transient overexpression, approximately 7.5 × 10⁵ cells were lipofected with plasmids coding for Myc-DDK-tagged versions of putative interactors (Fxyd6 (Origene: RC210607), Bst2 (Origene: RC207540) and Tmed1 (Origene: RC200255)). Twenty-four hours later, cells were pulsed with 5 µM pacSph and UV irradiated, followed by click-chemistry-based coupling of biotin and pull-down. For experimental verification of endogenous SL–protein interactions, approximately 4 × 10⁶ cells were used per sample.
For data analysis, band intensities (background corrected) were determined using the ImageLab software (Bio-Rad). For data analysis, band intensities (background corrected) were determined using the ImageLab software (Bio-Rad). Intensities from eluate bands were normalized to the intensities from the respective input band.

The intensities of bands corresponding to the RBD proteins were measured using Gel Doc 2000 and Image Lab software (Bio-Rad, Hercules, CA, USA) in order to measure the protein expression levels. Briefly, the blots were acquired using the Gel Doc 2000 apparatus; the images were imported into Image Lab software (Bio-Rad, Hercules, CA, USA); the contrast was adjusted such that the bands were clearly visible on the blot image; the area around each band was selected; the background intensity was subtracted from the blot image; the bands were then selected by drawing a tight boundary around them; the intensities of the selected bands were exported in an excel file format, which was used to perform further analyses.

Samples in different groups were lysed by radio immunoprecipitation assay lysis buffer (RIPA; Beyotime, China) containing protease and phosphatase inhibitor cocktail (Roche, Indianapolis, IN, USA). Proteins of the same quantity (30 μg/lane) in each group were separated by 10% or 15% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Temecula, CA, USA). Then, specimens were blocked by 10% skimmed milk in TBST at room temperature for 2 hours. Afterward, they were incubated with rabbit anti-SOD1 (1 : 1000, CST, USA), rabbit anti-SOD2 (1 : 1000, CST, USA), rabbit anti-PTEN (1 : 1000, CST, USA), rabbit anti-Phospho-Akt (1 : 1000, CST, USA), rabbit anti-Akt (1 : 1000, CST, USA), rabbit anti-Phospho-mTOR (1 : 1000, CST, USA), rabbit anti-mTOR (1 : 1000, CST, USA), mouse anti-GAPDH (1 : 1000, Santa Cruz Biotechnology, CA, USA), or mouse anti-β-actin (1 : 1000, Santa Cruz Biotechnology, CA, USA) antibodies overnight at 4°C. Secondary anti-mouse and anti-rabbit horseradish peroxidase- (HRP-) conjugated antibodies (1 : 10000, Boster, Wuhan, China) were used to conjoin the primary antibodies at room temperature for 2 hours. Finally, blots were visualized by electrochemiluminescence (ECL) substrate (ThermoFisher, USA). Signals were detected by Image Lab™ software (Bio-Rad, California, USA) and analyzed by Image Lab™ software (Bio-Rad, California, USA).