LC3A/B (D3U4C) XP® Rabbit mAb #12741

Protocol tips

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	- Make 1:1000 dilution of Primary antibody

Protocol tips
- Make 1:1000 dilution of Primary antibody

Publication protocol

Western blot was carried out according to the method previously reported by Chakrabarti and Ray (2015) with minor modification [27]. Both A172 and SR cells were lysed in the lysis buffer (50 mM Tris-HCl, pH7.4, 1 mM PMSF, and 5 mM EGTA) and homogenized by sonication after exposure to different concentrations of silibinin for 48 h. Then the whole cell lysates were centrifuged at 16,000g at 4°C for 30 min and the supernatants were used to prepare protein samples. Protein concentrations of the samples were quantified by the modified Bradford method after staining with Coomassie Plus protein reagent (Pierce Biotechnology, Rockford, IL). Protein samples (30 μg) were resolved by 4–20% gradient SDS-PAGE and transferred to the polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA). After blocking in 5% nonfat milk for 1 h, PVDF blots containing the resolved proteins were subsequently probed with a primary antibody (1 : 1000) against PARP, cleaved PARP, caspase 3, β-actin, LC-3 I/II, P62, mTOR, p-mTOR (Ser2448), YAP, p-S6k (Thr389), and p-4E-BP1 (Thr37/46) (Cell signaling Technology, Danvers, MA) overnight at 4°C. After washing with TBS for five times, the blots were further incubated with horseradish peroxidase conjugated goat anti-mouse or anti-rabbit IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 1 : 3000 dilution for 60 min to detect the primary antibody. Then, blots were thoroughly washed with TBS for five times and incubated with ECL reagents and exposed to X-OMATAR films for autoradiography. Autoradiograms were scanned using Canon CanoScan 9000F Mark II Photo Scanner

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for LC3A/B (D3U4C) XP® Rabbit mAb #12741 below.

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