Fig. 1.

Detection of endogenous human Usp27x. A, Schematic representation of the human Usp27x locus, Usp27x protein domains and the used human and mouse Usp27x variants. The human Usp27x locus (hUsp27x, NG_053124.1) is shown with the predicted/annotated ATG start codon which results in a 438 amino acid (aa) human Usp27x (named hUsp27x_S in this study) protein variant (A6NNY8, NCBI RefSeq.: NP_001138545.1). Of note, the murine Usp27x is predicted to also start with an ATG resulting in an Usp27x variant of the same size and domain structure (438aa, named mUsp27x_S in this study; Q8CEG8, NCBI RefSeq.: NP_062334.2). CTG indicates the alternative discovered start site [24] of human Usp27x which results in a protein with an 198aa extension at the N-terminus encoding a ZnF domain, absent in Usp27x_S. This human Usp27x variant (named hUsp27x_L in this study) is predicted to encode a protein of ~72 kDa (636 aa, [24]). Both Usp27x variants share the catalytic USP domain containing the UCH. The critical cysteine at position 87 (C87 in h/mUsp27x_S) for the active site corresponds to the position C285 in hUsp27x_L [20]. The last 19 aa (KQVLEHESEKVKEMNTQAY) present in both human Usp27x variants were used in this study as a target peptide sequence for the generation of the custom-made hUsp27x-antibody. B, Detection of endogenous human Usp27x by Western blot with the custom-made hUsp27x antibody confirms a variant of Usp27x that is larger than the predicted annotated form in various tested human cell lines. Endogenous human Usp27x of whole-cell-lysates from wild-type (WT), control (CTRL) or polyclonal Usp27x-deficient (Usp27x-2 and Usp27x-3) WM1158 melanoma or A549 lung cancer cells, as well as WT or single clone Usp27x-deficient CaCo2 (Usp27x-deficient clone 1/5) or 293FT cells (Usp27x-deficient clone 2/10) were detected by custom-made Usp27x-antibody via Western blotting. Arrow-heads indicate the specific signal for human Usp27x, asterisks indicate unspecific signal (n = 3). C, The custom-made hUsp27x-antibody is able to detect hUsp27x_S, but only detects hUsp27x_L at the endogenous level by Western blotting and immunoprecipitation. Endogenous human Usp27x from WT and single clone (clone 1/5) Usp27x deficient (Usp27x-KO) CaCo2 colon carcinoma lysate (800 µg) was immunoprecipitated with 4 µg of the custom-made hUsp27x antibody. The eluate was blotted along with 100 µg input, 100 µg IP-unbound flow through faction (IP unb.) and 10 µg lysate from 293FT cells transiently transfected with hUsp27x_S and 5 µg lysate from 293FT cells transiently transfected with hUsp27x_L (Con.). Black arrow-heads indicate specific signal for hUsp27x_L, grey arrow-heads indicate expected signal of the predicted hUsp27x_S, asterisks indicate unspecific signal in input and eluate (n = 3 for the IP, n = 2 for transient transfected controls)