Pvdf blot

Equivalent amounts of total cellular lysates (40 μg per treatment) were separated by 10–12% of SDS-PAGE gels, then transferred to the polyvinylidene fluoride (PVDF) blots (Merck Millipore, Darmstadt, Germany). After blocking in 10% non-fat milk, the blots were incubated with the applied primary antibodies, followed by incubation with corresponding secondary antibodies. Antibody-antigen binding was detected by an enhanced chemiluminescence (ECL) substrate kit (Invitrogen), with the results quantified by an ImageJ software (NIH, Bethesda, MD).

In brief, the protein lysates, from human tissues or cultured cells, were separated by 10–12% SDS-PAGE gels (40 μg protein in each lane), and transferred to polyvinylidene difluoride (PVDF) blots (EMD Millipore, Shanghai, China). The blots were blocked and incubated with the applied primary and secondary antibodies, with antibody–antigen binding examined by an ECL kit (GE Healthcare, Chicago, IL, USA). The same set of lysates were run in sister gels to test different proteins. The ImageJ software was utilized for data quantification.

The detailed protocols of Western blotting were previously reported [30 (link), 31 (link)]. In brief, lysate proteins were separated by SDS-PAGE gels [32 (link)], transferred to PVDF blots(Millipore, Shanghai, China). The blots were blocked and incubated with the designated primary and secondary antibodies. An ECL reagent kit (Pierce, Shanghai, China) was applied to detect the protein band under X-ray films. Data quantification was carried out by an ImageJ software (NIH). For the co-IP studies, the quantified protein lysates (1, 000 μg for each treatment) were pre-cleared and incubated with anti-Keap1 antibody [33 (link)]. Keap1-Nrf2 complex was captured by the G-Sepharose (“Beads”, Sigma), tested by Western blotting. Testing nuclear fraction lysates was described in our previous studies [30 (link), 31 (link)]

Lysate proteins were separated by 8-12% of SDS-PAGE gels, which were then transferred to PVDF blots (Millipore, Wuxi, China). After blocked in 10% milk, the blots were incubated with the designated primary and corresponding secondary antibodies. The protein signals were visualized via the ECL detection kit. Quantification of the signal was performed via the Image J software.

OS cells, with the applied genetic modifications, were harvested using the described lysis buffer [32 (link)]. Twenty μg lysate proteins per sample were separated by 10- 15% SDS gels, and transferred onto PVDF blots (Millipore, Shanghai, China). The blots were blocked, probed with applied primary and second antibodies [32 (link)]. The enhanced chemiluminescence (ECL) detection system was applied to visualize the targeted protein bands (based on the molecular weights), using x-ray films. For all the Western blotting assays, each lane was loaded with exact same amount of quantified protein lysates, then the same set of lysate samples were run in parallel (“sister”) gels. The ImageJ software was utilized for data quantification [33 (link), 34 (link)].

Spinal cord neurons were first trypsinized and washed. Neurons were then incubated with the cell lysis buffer (Biyuntian, Wuxi, China). Protein concentration was determined using the Bio-Rad protein assay kit (Shanghai, China). For each single treatment, exact 30–40 μg total lysate proteins (per lane) were separated by the SDS-PAGE gels (7.5–10%) [52 (link), 53 (link)], which were then transferred to the PVDF blots (Millipore, Suzhou, China). After blocking, the blots were incubated with designated primary and secondary antibodies. To visualize the targeted bands, the enhanced chemiluminescence (ECL) reagents (Pierce) were added, and X-Ray film development was employed.

The cell lysis buffer was purchased from Biyuntian (Wuxi, China). Quantified 30 μg of proteins from total cell lysates were separated by SDS-page gels (10–12%), which were transferred onto polyvinylidene difluoride (PVDF) blots (Millipore, Shanghai, China). After blocking, specific primary and corresponding secondary antibodies were added. The detection of the interested band was through the Enhanced chemiluminescence (ECL) reagents (Amersham Bioscience, Freiburg, Germany) and X-Ray film development. The ImageJ software was applied to quantify the intensity of each band.