FIGURE 2:

Ubiquitination occurs in the juxtamembrane region of Stx3. (A) Schematic representation of Stx3 wild-type and mutant constructs used for expression. Residues in the polybasic juxtamembrane region (PB) of wild-type Stx3 are shown. Two myc-epitope tags (white circles) and one His6 tag (black circle) were added to the COOH termini; PB, polybasic; TM, transmembrane domain. (B) HEK293T cells were transiently transfected with the indicated constructs, plated for 24 h and incubated with or without ALLN. Lysates were subjected to IP with anti-myc antibody and analyzed by IB with anti-Ubi. Bottom: Total lysates used for IP blotted with anti-myc antibody. Cells without construct transfection (lane C) serve as a negative control for the IP. (C) Sequence alignment of the juxtamembrane region of human plasma membrane syntaxins 1A, 2, 3, and 4, SwissProt accession numbers Q16623, P32856, Q13277, and Q12846 respectively. (D) Stx3 lysine mutant constructs used in this study (region 241-268 is shown). Lysine residues mutated to arginine are in bold letters. (E) Anti-myc Western blot of HEK293T treated with and without ALLN transiently expressing lysine mutants subjected to anti-myc IP. (F) Two different stable clones of DOX inducible MDCK cells stably transfected for Stx3-5R, c14, and c21, were subjected to IP with anti-myc antibody and IB with antibodies against Ubi, SNAP-23, and munc18b. (G) Immunocytochemistry of DOX-induced Stx3, Stx3-5R, or Stx3-6R (green) expresses in polarized MDCK cells cultured on Transwell filters. Scale bar: 10 μm.