Anti t7 antibody

Purified human proteasomes (~30 nM) were incubated with RPN13 (~300 nM), Ub_n–Sic1^PY (~300 nM) in degradation buffer (50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂ and 5 mM ATP) at 37 °C. Purified recombinant USP14 variants (~1.2 μM) were incubated with proteasome for 20 min at room temperature before initiating the degradation reaction. The reaction mixtures were incubated at 37 °C for 0, 0.5, 1.0 and 2.0 min, or 10 °C for 0, 0.5, 1.0, 5.0, 10 and 30 min, then terminated by adding SDS loading buffer and subsequently analysed by western blot using anti-T7 antibody (Abcam, 1:1,000 dilution), which was used to examine fusion protein T7–Sic1^PY.

Erlotinib was purchased from MedChem Express. Cetuximab was from MERCK Serono. Trametinib and vemurafenib were from ApexBio. Selumetinib and MG132 were from Selleckchem. Recombinant human IL-36γ (catalog 6835), mouse IL-36γ (catalog 6996), and human IL-36Ra (catalog 1275) were from R&D Systems. Pam3CSK4 was from InvivoGen. The goat anti–IL-36γ (catalog AF2320) and anti-mouse KLF4 (catalog AF3158) antibodies were from R&D Systems. The rabbit anti–IL-36γ (catalog LS‑C201142) and its blocking peptide (catalog LS-E45854) were from LifeSpan BioSciences. The anti–human KLF4 antibody (catalog AM09057PU-N) was from Acris. The anti–β-actin (catalog A5441), anti-FLAG (catalog F1804), and anti-myc (catalog C3956) antibodies were from Sigma-Aldrich. The anti–human KLF4 (catalog 12173), anti-ERK (catalog 9107), anti–phospho-ERK (catalog 4370), and anti–phospho-threonine/proline (catalog 9391) antibodies were from Cell Signaling Technology. The anti-T7 antibody was from Abcam (catalog ab9138). The anti-HA antibody was from Santa Cruz Biotechnology (catalog sc-805). The secondary antibodies used were alkaline phosphatase–conjugated mouse IgG (catalog S372B), rabbit IgG (catalog S373B), and goat IgG (V115A) from Promega. Live C. acnes was prepared as previously described (63 (link)).