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. 2022 Feb 16;14(3):e14901. doi: 10.15252/emmm.202114901

Figure EV2. Diminished OTULIN protein expression in patient‐derived cells is not due to reduced mRNA expression or increased degradation via the proteasome or the lysosome.

Figure EV2

  1. OTULIN protein is depicted.
  2. OTULIN antibodies by Abcam (antigen corresponding to AA 60–158) or by Cell Signaling (recombinant fragment surrounds S76 without spanning M86) were tested in parallel on A549 OTULIN KO cells transfected with different OTULIN constructs as indicated. One representative of two independent experiments is shown.
  3. Relative mRNA expression of OTULIN in fibroblasts and B cells with two different primer pairs is depicted. Data are presented as mean ± SD of six independent experiments; dots represent individual experiments performed in three technical replicates; ns, non‐significant, unpaired t‐test.
  4. A549 OTULIN KO cells were transfected with the different OTULIN constructs as indicated. The following day, cells were treated with 50 µg/ml cycloheximide (CHX) for the indicated times, harvested and analyzed by Western blot for OTULIN protein expression. Tubulin served as loading control. One representative of three independent experiments is shown.
  5. A549 OTULIN KO cells were transfected with the different OTULIN constructs as indicated. The following day, cells were treated with 50 µg/ml CHX alone or in combination with 1 µM Bortezomib (BTZ) or 1 µM Bafilomycin A1 (Baf A1) for 8 h or left untreated (DMSO). Expression of the proteins indicated was analyzed by Western blot. One representative of three independent experiments is shown.