His 1

Rabbit polyclonal antibodies raised against TSEN2 (N-NGDSGKSGGVGDPREPLG-C), TSEN34 (N-QASGEQEEAGPSSSQAGPSNG-C), and TSEN54 (N-RSRSQKLPQRSHGPKDFLPD-C)^{23 (link)} (Gramsch Laboratories, Schwabhausen, Germany) were affinity-purified from rabbit sera and eluted sequentially by addition of 1.5 M MgCl₂ and 0.1 M glycine, pH 2.5. Eluates were dialyzed against HEPES-buffered saline (HBS, pH 7.9) overnight, supplemented with 10% (v/v) glycerol, and stored at −80 °C. Small-scale pilot experiments were set up to assess the suitability of the affinity-purified antibodies for immunoprecipitation experiments using total cell lysate from HEK 293 cells. Antibodies used in western blotting (WB) and immunoprecipitation (IP) were: anti-TSEN15 (rabbit polyclonal, Atlas Antibodies, HPA029237; WB dilution 1:1000), anti-TSEN34 (IP, WB dilution 1:5000), anti-TSEN54 (WB dilution 1:5000), anti-TSEN2 (IP, WB dilution 1:5000), anti-GAPDH (rabbit monoclonal, 14C10, Cell Signaling Technology, #2118; WB dilution 1:1000), anti-β-actin (mouse monoclonal, clone AC-74, Sigma-Aldrich, A2228; WB dilution 1:3000), anti-mouse IgG–peroxidase conjugate (secondary goat polyclonal, Sigma-Aldrich, A2554; WB dilution 1:20.000), anti-rabbit IgG–peroxidase conjugate (secondary goat polyclonal, Sigma-Aldrich, AP307P; WB dilution 1:20.000), anti-polyHistidine (mouse monoclonal, clone HIS-1, Sigma-Aldrich, H1029; IP).

Recombinant HIS-tagged proteins were produced by IPTG induction (0.4 mM) of T7 Express lysY/I^q Competent E.coli (New England Biolabs C3013I) transformed with HIS-tag expressing control vector, pET-30a+ (Novagen) or HIS-tagged KRT76 domains in pDEST17 gateway backbone (Life Technologies), grown for 6–8 hours at 37°C in low salt LB, supplemented with 100 µg/ml ampicillin or 50 µg/mL kanamycin (as required). Recombinant protein was purified using 0.1 ml per 1 ml of culture of PopCulture lysis reagent (Novagen), 1 µl per mL of culture of 40 U/ml of Lysonase bioprocessing reagent (Novagen), protease inhibitors (Sigma P8849), and His-Mag beads (Novagen) according to manufacturer's protocols. Bound recombinant HIS and HIS-KRT76 protein were washed and stored at 4°C as a 1∶2 resin slurry in Tris-saline pH 7.4 containing protease inhibitors. Paw pad skin of adult was collected in RIPA lysis buffer and incubated with HIS or HIS-KRT76 overnight at 4°C. HisMag bead-bound HIS and HIS-KRT76 + lysates were then washed four times in Tris-saline pH 7.4 including 1% Triton X-100 and immunoblotted for mCLDN1 (Santa Cruz Biotechnology, SC-81796) and the HIS tag (Sigma-Aldrich, clone HIS-1).