Trypsin kit

Equal weights of heart tissues from three control mice or three SIMD mice were mixed and homogenized with RIPA lysis buffer containing a protease inhibitor. Total cell extracts were centrifuged at 18,000 g for 15 min at 4°C. The supernatant fractions were immunoprecipitated using anti-DDB1 and anti-DCAF8 antibodies. The purified protein complexes were resolved by a 10% SDS-PAGE gel and then stained with a Pierce silver stain kit (Thermo Fisher Scientific, #24612). The stained protein bands were cut into ~0.5 mm small slices, followed by digestion with a Trypsin Kit (Thermo Fisher Scientific, #60109101) and mass spectrometry analysis according to a previous protocol 25 (link).

Cells (1×10⁸) expressing pCDNA3-2×Flag-3×HA-c-Myc or pCDNA3-2×Flag-3×HA (Empty vector, control) were lysed in RIPA lysis and extraction buffer (Thermo Fisher Scientific, #89900) containing 1 × protease inhibitor cocktail (Sigma-Aldrich, #P8340). Cell extracts were centrifuged at 14,000 rpm for 30 min and the supernatant was incubated with anti-Flag agarose (Sigma-Aldrich, #A2220) to pull down Flag-associated proteins at 4°C overnight. The resulting Flag-associated proteins were washed five times with RIPA lysis and extraction buffer and then eluted with 100 μg/mL Flag peptide (Sigma-Aldrich, #F4799). The obtained protein complex was further incubated with anti-HA agarose (Thermo Fisher Scientific, #26181) to pull down HA-associated proteins at 4°C overnight. The resulting HA-associated proteins were washed five times with RIPA lysis and extraction buffer, followed by loading onto a 10% SDS-PAGE gel for separation. The gel was subsequently performed sliver staining with a kit (Thermo Fisher Scientific, #24612). Protein bands were cut into small slices and then digested with a Trypsin Kit (Thermo Fisher Scientific, #60109101). Mass spectrometry analysis was performed to determine c-Myc-associated proteins following a protocol as described previously 27 (link).

The degenerative IVD tissue (0.1 g) from an IDD patient under Pfirrmann grade IV was homogenized in 1 mL RIPA buffer containing the protease inhibitor. The supernatant of the cell lysate was immunoprecipitated using anti-Smad4- and IgG-coupled protein A beads (Santa Cruz Biotechnology, Shanghai, China; #sc-2001). The enriched proteins were rinsed five times with PBST buffer and then loaded onto a 12% SDS-PAGE gel for separation, followed by staining with the ProteoSilver Kit (Sigma-Aldrich; #PROTSIL2). The positive bands were cut into small pieces and then digested using the Trypsin Kit (Thermo Fisher Scientific; #60109101). The eluted proteins were subjected to MS analysis.
The co-IP assay was performed as described previously 25 (link). In brief, different combinations of plasmids, including pCDNA3-2×Flag + pCDNA3-Myc, pCDNA3-2×Flag + pCDNA3-Myc-HDCA1, pCDNA3-2×Flag-Smad4 + pCDNA3-Myc-HDCA1, and pCDNA3-2×Flag-Smad4 + pCDNA3-Myc were co-transfected into HNPC cells. After 48 hours of transfection, cells were subjected to the IP procedure using anti-Flag agarose (Sigma-Aldrich; #A4596) and anti-Myc-agarose (Sigma-Aldrich; #A7470). The enriched protein complexes were probed with anti-Flag (Abcam; #ab125243) and anti-Myc (Abcam; #ab32).