Phosphothreonine

Recombinant Rv2623 derived from M. smegmatis, as well as the native USP immunoaffinity-purified from M. tuberculosis and BCG cell lysates were analyzed for phosphorylation using a dot blot assay. Briefly, increasing concentrations of Rv2623 protein derived from the various sources were spotted onto nitrocellulose membrane. Phosphothreonine (Sigma-Aldrich; St. Louis, MO) served as positive control. The membrane was allowed to dry, followed by blocking with 5% BSA in Tris-HCl (20 mM Tris-HCl; pH 7.5) containing 150 mM NaCl, 0.05% Tween 20) (TBST). The membrane was probed with anti-Phosphothreonine antibodies: clone #42H4 mouse monoclonal or rabbit polyclonal antibodies (Cell Signaling Technology; Danvers, MA), washed thrice with TBST and then reacted with appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Sigma-Aldrich; St. Louis, MO). The blot was developed using ECL reagent (Amersham; Piscataway, NJ).

The generation of the 90K-myc and ISG15-V5 constructs was described previously [19 (link)]. HA-Ub was kindly provided by K.Y. Lee (Chonnam National University, Gwangju, Korea). The antibodies used in the present study were as follows: anti-E-cadherin (C20820, BD Biosciences, San Jose, CA, USA; CS#3195, Cell Signaling Technology, Danvers, MA, USA), p120-catenin (610133, BD Biosciences), β-catenin (#9582, Cell Signaling Technology), actin (A2066, Sigma, St. Louis, MO, USA), myc (M047-3, MBL, Woburn, MA, USA), V5 (PM003, MBL), HA (H9658, Sigma), tubulin (sc-5286, Santa Cruz Biotech, Dallas, TX, USA), phospho-serine (P3430, Sigma), phospho-tyrosine (05-321, Upstate, Molsheim, France), phospho-threonine (P3555, Sigma), phospho-Tyr-228–p120-catenin (ab32403, abcam, Cambridge, MA, USA).