Cells (5 × 106) were lysed for 20 min with lysis buffer (Beyotime Biotechnology) containing protease inhibitors (Roche, Indianapolis, IN, USA). The protein concentrations were determined by the BCA method (Pierce, Thermo Fisher Scientific Inc., Rockford, IL, USA). After centrifugation at 16,400×g for 15 min at 4 °C, the samples were resolved by SDS/PAGE, transferred to PVDF membranes (Immobilon-P membrane, Millipore, Massachusetts, USA), and analysed by immune blotting using HRP-conjugated secondary antibodies. The membranes were blocked with 5% (wt/vol) skimmed milk in TBS plus Tween 20 at 4 °C overnight before probing with antibodies. Information on the antibodies are provided in Supplementary Table 3 . An enhanced chemiluminescent (ECL) chromogenic substrate was used to visualize the bands (Pierce). Visualization was performed using the Enhanced Chemiluminescence Plus Western Blotting Detection System (GE Healthcare, Connecticut, USA) and LAS-4000EPUV mini Luminescent Image Analyzer (GE Healthcare). The uncropped blots are shown in Supplementary Fig. 14 .
Las 4000epuv mini luminescent image analyzer
Manufactured by GE Healthcare
Sourced in United States
The LAS-4000EPUV mini Luminescent Image Analyzer is a compact, versatile imaging device designed for detecting and analyzing luminescent signals. It can capture high-quality images of a variety of samples, including gels, membranes, and microplates. The instrument utilizes a UV light source and a high-sensitivity CCD camera to generate detailed images of luminescent samples.
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2 protocols using las 4000epuv mini luminescent image analyzer
1
Western Blot Analysis of Protein Samples
2
Western Blot Analysis of CAL-62 Cells
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We used lysis buffer (YEASEN, Shanghai, China) with protease inhibitors (Roche, Indianapolis, IN, USA) to lyse CAL-62 cells. Protein concentrations were determined by the BCA method (Pierce, Therrmo Fisher Scienti c Inc., Rockford, IL, USA). The protein samples were subjected to SDS/PAGE and transferred to PVDF membranes (Immobilon-P membrane, Millipore, Massachusetts, USA). The membranes were blocked with 5% skimmed milk in TBS plus Tween 20 at room temperature for 1 hour, followed by incubation with target antibodies at 4 °C overnight. Information on the antibodies are provided in Table S3. After incubation with HRP-conjugated secondary antibodies for 1 hour, visualization of the protein bands was achieved by an enhanced chemiluminescent chromogenic substrate using the Enhanced Chemiluminescence Plus Western Blotting Detection System (GE Healthcare, Connecticut, USA) and LAS-4000EPUV mini Luminescent Image Analyzer (GE Healthcare).
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