Fig 2: Downregulation of ULF and SIVA1 proteins inhibits ubiquitination and degradation of p14ARF protein in H. pylori-infected cells.(A) Analysis of p14ARF protein expression after downregulation of ULF (left panel), SIVA1 (central panel), or MKRN1 (right panel) with specific siRNAs in SNU1 cells co-cultured with H. pylori strains 7.13 or B128 for 4hrs or left uninfected. Transfections with scrambled siRNA (Scr siRNA) were used as controls (n = 3). (B) The same as (A), but SNU1 cells were transfected with plasmids expressing ULF (left panel) or SIVA1 (right panel) proteins. Transfections with empty vector (pcDNA3) were used as controls (n = 3). (C) Analysis of p14ARF protein ubiquitination after downregulation of ULF or SIVA1 with siRNAs in SNU1 cells co-cultured with H. pylori strain 7.13. Bottom panels show expression of ULF and SIVA1 in input cell lysates. All experiments were performed at least three times (n = 3) with representative blots shown. Graph panels depict the densitometric analyses. Protein expression levels in control cells were arbitrarily set at 1.

Fig 3: Analyses of ULF and SIVA1 protein expression in the murine stomach.(A) A representative image showing IHC staining for SIVA1 protein in the corpus and antrum of H. pylori-infected and non-infected mice (n = 6/group). (B) The same as (A) but ULF protein staining is shown. (C) The same as (A and B), but Western blotting was used for analyses of ULF and SIVA1 proteins in gastric tissue homogenates. Bottom panel shows the densitometric analyses. (D) Protein expression of ULF (left panel) and SIVA1 (right panel) in gastric organoids derived from the antropyloric region of the murine stomach that were infected with H. pylori strain 7.13 in vitro or left uninfected. Panels show representative light microscopic and immunofluorescence images. Bottom panel shows quantification of ULF and SIVA1 fluorescence intensity/cell. Data were analyzed using unpaired 2-tailed t-test. Data are displayed as mean ± SD.

Fig 4: Trip12 modulates mRNA levels of fiber-type-specific genes in differentiating C2C12 cells. C2C12 cells were transfected with siRNA for either EGFP (negative control) or Trip12, and medium was switched to DM 24 h after transfection to induce differentiation into myotubes. Total RNA was then extracted every 24 h, and the time course of mRNA levels of Trip12, Myh4 (MyHC-IIb), Myh7 (MyHC-I/β), and myogenin (Myog) were quantified by RT-qPCR using Huwe1 and Tbp as reference genes (see the Methods section for details). Data are normalized for those from EGFP siRNA-transfected cells and represented as mean ± SD (n=3). No expression change between EGFP siRNA- and Trip12 siRNA-transfected samples is expressed as 1 on the graph. *p < 0.05, **p < 0.01, ***p < 0.005.

Fig 5: SOX6 coiled-coil domain physically interacts with TRIP12 HECT domain. (A) Schematic representation of the human SOX6 protein [17]. The coiled-coil (CC) domain containing the leucine-zipper (LZ) motif and Q-box (aa 143–304) was used as bait for screening a human heart cDNA library. The amino acid sequence of the CC domain is 100% conserved between mice and humans. (B) Schematic representation of the full-length human TRIP12 protein. The partial TRIP12 cDNA clone identified by yeast hybrid screening contained the most C-terminal HECT domain (aa 1614–2040). (C) Diagrams of the bait and prey expression vectors used for Co-IP. The numbers indicate the amino acid regions used for each vector construct. The SOX6 CC-domain was tagged with c-Myc at the C-terminus. The TRIP12 HECT domain was tagged with HSV at the C-terminus. (D) Co-IP assays were performed using HEK293 cell lysate transfected with the bait and prey expression vectors depicted in (C). Input lanes contained 2% (10 μg) of un-manipulated lysate. Rabbit polyclonal antibodies used for pull down are listed under IP. Mouse monoclonal antibodies used for Western blot (WB) are indicated below each panel. GST antibody was used as a negative control.