Anti ubiquitin antibody ab7780

siNC or siTRIM27 was transfected. Cells were lysed in RIPA buffer (1% SDS) by sonication. Lysates were incubated with Protein A/G PLUS-agarose (sc-2003; Santa Cruz Biotechnology, USA) for 1 h. IgG (sc-2027; Santa Cruz Biotechnology, USA) was added to all samples and incubated overnight at 4 °C. Nuclear pellets were collected following centrifugation at 3000 rpm at 4 °C, followed by washing with Protein A/G PLUS-agarose beads four times. Proteins were loaded onto 4–20% gradient SDS-PAGE gels. Signals were detected by anti-IκBα antibody (#4812; Cell Signaling Technology, Danvers, MA, USA) and anti-ubiquitin antibody (ab7780; Abcam, UK).

An anti-PDCD4 antibody was prepared by immunizing rabbits with a synthetic peptide corresponding to the N-terminal amino acid sequence [12 (link)]. This antibody was used for the Western blotting analyses. Antibodies against β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-PDCD4 (Human) polyclonal antibody (pAb) (PD024), guinea pig anti-p62 C-terminal pAb antibody (PM066), rabbit anti-p62 (SQSTM1) pAb antibody (PM045), rabbit anti-Atg5 pAb antibody (PM050), and mouse anti-LC3 monoclonal antibody (mAb) (M152-3) were obtained from MBL (Tokyo, Japan). Anti-ubiquitin antibody (ab7780) and donkey anti-mouse I_gG H&L (DyLight650) antibody (ab96878) were purchased from Abcam (Cambridge, UK). Alexa Flour 488 donkey anti-rabbit I_gG (H+L) antibody was obtained from Thermo Fisher (Waltham, MA, USA). Alexa Flour 555-conjugated donkey anti-guinea pig I_gG (H+L) antibody (bs-0358D-A555) was obtained from Bioss Antibodies Inc. (Woburn, MA, USA). Anti PDCD4 mouse monoclonal antibody (sc-376430) was obtained from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA). The antibodies were used according to the protocols provided by the respective companies.

To detect ubiquitinated-PCBP1, TPC1 cells were transiently transfected with HA–PCBP1 or FLAG–UBE4A as indicated. For immunoprecipitation (IP), whole cell lysates were prepared as above. Cell lysates (2000 μg) were incubated for overnight at 4°C with 50 μg of either mouse anti-HA antibody or mouse IgG (Sigma-Aldrich, Shanghai, China) cross-linked to protein A/G beads (Pierce Crosslink IP Kit, Life Technologies, Shanghai, China). The immune complexes were collected by centrifugation, washed five times in IP lysis buffer, and eluted with glycine. The eluant were resolved by SDS/PAGE and probed by anti-Ubiquitin antibody (#ab-7780, Abcam).