Protein g hrp conjugate

Human cDNAs coding for FMNL2 (Q96PY5-3) and FMNL3 (Q8IVF7-3) were purchased from Promega (Madison, WI, USA). ECL Prime Western Blotting Detection Reagent was sourced from GE Healthcare (Amersham, UK). The dye terminator cycle sequencing kit, Lipofectamine LTX with PLUS reagent and Hoechst 33342 were obtained from Life Technologies Corp. (Carlsbad, CA, USA). Anti-FLAG monoclonal antibody and anti-mouse IgG-FITC antibody were obtained from Sigma (St. Louis, MO, USA). Tetradec-13-ynoic acid (Alk-Myr) was sourced from Cayman Chemical Co. (Ann Arbor, MI, USA). 5-TAMRA Azide (Az-TAMRA) was purchased from Click Chemistry Tools (Scottsdale, AZ, USA). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) were obtained from Sigma (St. Louis, MO, USA). Protein G-HRP conjugate was sourced from Bio-Rad (Hercules, CA, USA). HRP-conjugated anti-mouse IgG was purchased from Cell Signaling Technology (Danvers, MA, USA). The other reagents used were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) or Daiichi Pure Chemicals Co., Ltd. (Tokyo, Japan), and were of analytical or DNA grade.

In order to validate human IgG binding to different M1 protein regions ELISA was used. The recombinant, full-length M1 protein and the synthetic peptides from ProteoGenix (see above; all 10 μg ml⁻¹) were immobilized on MaxiSorp 96-well ELISA plates (Thermo Fisher Scientific) overnight at 4 °C. The plates were washed three times with 1 × PBST, and blocked with 2% BSA in 1 × PBST for 1 h at 37 °C. The plates were washed again three times with 1 × PBST, and IVIG (4 mg ml⁻¹) was added as a two-fold dilution series. The plates were incubated 1 h at 37 °C, washed three times with 1 × PBST and affinity-purified protein G HRP conjugate (Bio-Rad, catalog number 170-6425) diluted 1:3000 in 1 × PBST was added to the wells. The plates were incubated 1 h at 37 °C, washed three times with 1 × PBST and color developed with 2,2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS; Sigma) for 5 min at RT in the dark, prior to determining the absorbance at 415 nm. The GFP-based peptide was used as a negative control in the assays, and its absorbance values were subtracted from the experimental data prior to analysis in Prism version 8.0.2 (GraphPad). Data analysis used two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests. Statistical significance levels were set at P < 0.0332, P < 0.0021, P < 0.0002 and P < 0.0001.

To analyze SUMOylation within cells, we used Lipofectamine 2000 to transfect HeLa cells (ATCC CRL-2, Manassas, VA) with plasmids encoding wild-type HA-TGM2 along with either SUMO1, SUMO2 and SUMO3 plasmids respectively. Cells were selected and grown according to a previously described protocol [28 (link)]. Cell lysates were immunoprecipitated with a HA-specific affinity matrix gel (Roche, Pleasanton, CA), after which immunoprecipitate was washed in lysis buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1 mM DTT, 10% glycerol, 10 mM EDTA), followed by elution and proteins resolved by SDS-PAGE and immunoblotted using TG2 antibody, RanGAP antibody (Cell Signal, cat# 36067), and protein G HRP-conjugate (BioRad, Hercules, CA).