Protein a g

Bovine MDSCs were lyzed in 1 ml of RIPA buffer after differentiation. Then, 20 μl of protein extract was used as an input. The remaining portion was divided equally into two equal samples, to which 2 μg of rabbit IgG (Beyotime Biotechnology Beijing, China) and indicated antibodies were added at 4°C overnight. Subsequently, protein A + G (Beyotime Biotechnology) was incubated with the samples at 4°C for 3 h. After centrifugation, the beads were washed five times with RIPA buffer, and the harvested beads were resuspended in the same volume of 5 × SDS sample buffer and boiled for 10 min to remove the Sepharose beads. Then, the immunoprecipitates were analyzed by western blotting. Next, the gel was soaked in Coomassie Brilliant Blue for 30 min, and the color was eluted overnight by acetic acid. The entire gel was sent to Applied Protein Technology (Shanghai, China) for mass spectrometry-based sequencing. Based on the sequencing results, a protein that may bind to podocan and exert biological functions was selected. The above procedure was carried out using a specific antibody against the selected protein, and a co-immunoprecipitation assay was used to precipitate the podocan protein.

U251 cells infected with PDCL3-3 knockdown lentivirus and its control lentivirus were collected, and their RNA was extracted for transcriptome sequencing (biological replicates were performed three times). Subsequently, the DEGs were screened out (|log2FC|>0.5 and q-value<0.05) and are shown with a heatmap. GO terms and KEGG analysis were used to annotate and explore potential functional mechanisms. For IP-MS, U251 cells infected with myc-PDCL3 were lysed in IP buffer containing 1% NP-40, 50 mM NaF, 2 mM Na3VO4, 4 mM Na pyrophosphate and protease inhibitors. A total of 3 μg of mouse anti-myc monoclonal antibody or IgG was added to the cell lysates, and 4 hours later, the samples were incubated with 30 μL Protein A/G (Beyotime, China) at 4°C overnight. The precipitates were washed 5 times with cold PBS and boiled for 5 min in 40 μL of 2x loading buffer (Biosharp, China), followed by SDS-PAGE and silver staining. Mass spectrometry and analysis were performed to identify the immunoprecipitation-enriched proteins.

The samples were lysed in RIPA buffer supplemented with protease inhibitors at 4°C for 30 min. The protein suspensions were incubated with an anti-SUMO-1 antibody (#4930; 1:50 dilution, Cell Signaling Technology) or control IgG (A7016, Beyotime, Shanghai, China) with gentle shaking at 4°C overnight, and then Protein A/G (P2012, Beyotime) was added and incubated with gentle shaking at 4°C for 1–3 h. Each mixture was centrifuged at 2,500 rpm for 5 min at 4°C. Subsequently, the supernatant was removed, and the beads were washed for four times using PBS. The immunoprecipitated protein were removed from the beads by heating in 1× sample loading buffer at 100°C for 10 min, and the samples were subjected to Western blot analyses using anti-α-syn antibody (#2642; 1:1,000 dilution, Cell Signaling Technology).

To directly quantify the enrichment of ubiquitinated TAZ in cell extracts, Ub plasmids, Myc-TAZ vectors and Flag-USP1 or Flag vectors were transfected into HEK293 cells. After 24 h, the cells were treated with 10 μM MG132 for 8 h. Then, total protein was extracted, and 20 μL of protein A + G (Beyotime, P2012) was used to preclear the lysate for 2 h. The supernatant was harvested and immunoprecipitated with an anti-TAZ antibody. Western blotting was performed with an anti-HA antibody to detect polyubiquitination of TAZ.

Co-IP was performed using the relevant antibodies and protein A/G-conjugated Dynabeads (Beyotime) according to the manufacturer's instructions. In brief, cell lysates were incubated with antibodies overnight at 4 °C. protein A/G-conjugated beads were added into the lysate at 4 °C for 2 h. Then, the beads were washed with lysis buffer or PBS and boiled in SDS loading buffer. Western blotting was used to detect the immunoprecipitated proteins.

With the protease inhibitors, protein samples were extracted in RIPA buffer. The mixtures were treated with anti‐Parkin antibody (1:50, Abcam, Cat. #ab77924) or control IgG (Beyotime, Cat. # A7016) at 4°C overnight followed by incubation with Protein A/G (Beyotime, Cat. #P2012) at 4°C for 2 hr. After the samples were centrifuged at 1,500 g for 5 min, the supernatant was removed. Subsequently, the beads were washed three times with PBS to remove the proteins. Western blot analyses were then performed using anti‐α‐syn antibody (1:1,000, CST, Cat. #4179).