Figure 3. USP28 regulates ∆Np63 protein stability in SCC tumour cell lines.

• A
  Immunoblot of endogenous USP25, USP28 and ∆Np63 immunoprecipitated (IP) from A‐431 cells and co‐precipitated ∆Np63 or USP28. Beads were coupled to specific USP25, USP28 and ∆Np63 antibodies or non‐specific rabbit IgG as control. The input corresponds to 10% of the total protein amount used for the IP (TUBULIN as loading control).
• B
  Inducible depletion of USP28 in A‐431 upon treatment with doxycycline (1 μg/ml) for 96 h, Western blot (left, VINCULIN as loading control) and qPCR analysis of USP28 and ∆Np63 expression relative to ACTIN (right) were performed.
• C
  Tandem ubiquitin binding entity (TUBE) pulldown of endogenous ubiquitylated ∆Np63 in A‐431 cells upon DOX depletion of USP28. Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization.
• D
  Cycloheximide (CHX) chase assay (100 μg/ml) of control or inducible sh‐USP28 A‐431 cell line (EtOH or 1 μg/ml dox) for indicated time points. Representative immunoblot (left, VINCULIN as loading control) of USP28 and ∆Np63 and quantification of relative protein abundance (right).
• E
  Doxycycline induced murine USP28 overexpression (EtOH or 1 μg/ml dox for 96 h) in A‐431 cells followed by immunoblot (VINCULIN as loading control) and qPCR analysis of USP28 and ∆Np63. For qPCR, human USP28 and murine USP28 (mUSP28) primers were used. Relative mRNA was calculated using ∆∆Ct analysis for human USP28 and ∆Ct for mUSP28 (ACTIN as housekeeping).
• F
  TUBE pulldown of endogenous ubiquitylated ∆Np63 in A‐431 cells upon overexpression of mUSP28 for 96 h (EtOH or 1 μg/ml dox). Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization.
• G
  CHX chase assay (100 μg/ml) of control or inducible mUSP28‐overexpressing A‐431 cell line (EtOH or 1 μg/ml dox) for indicated time points. Representative immunoblot analysis of USP28 and ∆Np63 as well as quantification of relative protein abundance (VINCULIN as loading control).
• H
  Immunoblot of control (sh‐NTC) and two independent shRNA targeting USP28 (sh‐USP28#1 and #2) for ∆Np63 and USP28 protein abundance in LUDLU‐1^adh (VINCULIN as loading control), followed by qPCR analysis of USP28 and ∆Np63 expression relative to ACTIN.
• I
  Doxycycline induced mUSP28 overexpression (EtOH or 1 μg/ml dox for 96 h) in LUDLU‐1^adh cells followed by immunoblot (VINCULIN as loading control) and qPCR analysis of USP28, mUSP28 and ∆Np63. Relative mRNA was calculated using ∆∆Ct analysis for human USP28 and ∆Ct for mUSP28 (ACTIN as housekeeping for the analysis).

Data information: Western blots shown are representative of three independent experiments (n = 3). All quantitative graphs are represented as mean ± SD of three experiments (n = 3). P‐values were calculated using two‐tailed t‐test statistical analysis; *P < 0.05; see also Appendix Fig S2 and Appendix Table S3 (exact P‐values and statistical test used).Source data are available online for this figure.