By examining the genomic sequence in these excision mutants, we confirmed that one mutant had a deletion in the transcript (Number S2A). the gene by alternative splicing. The shorter form consists of 1,058 amino acids and the longer form of 1,507 amino acids. The difference in length is due to an insertion of 449 amino acids after residue 225 in the long form. In these experiments, we used GFP-tagged shorter VprBP isoform. (D) Connection of Lgl with Mahjong protein. Myc-tagged Lgl was coexpressed with GFP or GFP-Mahjong in S2R+ cells, and immunoprecipitation was performed with anti-GFP antibody, followed by Western blotting with anti-Myc and anti-GFP antibodies. Mouse and rabbit anti-GFP antibodies were utilized for immunoprecipitation and Western blotting, respectively. The arrow and arrowhead indicate the positions of GFP-Mahjong and GFP, respectively.(0.45 MB TIF) pbio.1000422.s001.tif (444K) GUID:?BA4B4289-0537-493D-A6E8-8A427D667FCA Number S2: (A) PNU-176798 Schematic representation of the genome region containing the (CG10080) locus. Expected genes are indicated as boxes. The P-element insertion site of GT-000304 and the extent of the deletion are indicated. (B) A collection graph showing the growth defect of the homozygous mutant larvae and the trans-heterozygous mutant larvae with is definitely deleted. (C) Assessment of the heterozygous control female (remaining) with the homozygous mutant female rescued by manifestation of UAS-Mahjong under the control of actin-GAL4 (ideal). (D) Transverse sections of a wing disc of homozygous larva that were immunostained with anti-DE-Cadherin and anti-Dlg antibodies. (E) Wing discs with wild-type (lacking GFP) and GFP-expressing wild-type clones at 72 h, 96 h, and 120 h (remaining to ideal) after clone induction. (F) Transverse sections of a wing disc with heterozygous cells (expressing -gal, green) at 144 h after clone induction. (D, F, and G) Nuclei were stained with DAPI (blue). (F and G) Apoptotic cells were labeled with anti-cleaved Caspase-3 antibody (reddish).(1.06 MB TIF) pbio.1000422.s002.tif (1.0M) GUID:?74F702B8-C117-485C-A592-6FEEB4Abdominal17DF Number S3: Characterization of the effect of Mahjong knockdown in PNU-176798 MDCK epithelial cells. (A) Establishment of MDCK cell lines that stably communicate Mahjong shRNA inside a tetracycline-inducible manner. Parental MDCK or MDCK pTR Mahjong shRNA cells (clone 3 or 4 4) were cultured with or without tetracycline for 48 h, and cell lysates were analyzed by Western blotting with anti-Mahjong or anti-GAPDH antibody. Note that similar phenotypes were observed in cell polarity and cell competition with clones 3 and 4. (B) Effect of Mahjong knockdown on morphology in MDCK cells. MDCK pTR Mahjong shRNA cells were cultured with or without tetracycline for 48 h and were analyzed by phase-contrast microscopy. (C) Knockdown of Mahjong does not affect the manifestation of mLgl2 or PKC. Parental MDCK or MDCK pTR Mahjong shRNA cells were cultured with or without tetracycline for 48 h, and cell lysates were analyzed by Western blotting with anti-mLgl2 or anti-PKC antibody. (D and E) Immunofluorescence analyses of cell polarity markers in MDCK Mahjong shRNA cell cysts. MDCK pTR Mahjong shRNA cells were seeded in collagen gels PNU-176798 and incubated with or without tetracycline for 11 d. Immunostaining was performed with anti-PKC antibody and TRITC-labeled phalloidin (D) or with anti–catenin and anti-ZO-1 antibodies (E). (B, Rabbit Polyclonal to TSEN54 D, and E) Level bars: 10 m.(0.30 MB TIF) pbio.1000422.s003.tif (291K) GUID:?814B192C-E241-4FF0-8940-87AB7A5A1A48 Figure S4: Overexpression of Mahjong alleviates the cell competition phenotype in MDCK Mahjong shRNA cells. (A) Establishment of MDCK pTR Mahjong shRNA+GFP-Mahjong cells that stably communicate Mahjong shRNA and GFP-tagged human PNU-176798 being Mahjong inside a tetracycline-inducible manner. Because of mismatch of a base pair, manifestation of Mahjong shRNA does not knock down exogenously indicated human being Mahjong. MDCK pTR Mahjong shRNA+GFP-Mahjong cells were cultured with or without tetracycline for 48 h, and cell lysates were analyzed by Western blotting with anti-Mahjong or anti-GFP antibody. Arrowhead, arrows, and asterisks indicate the positions of endogenous Mahjong protein, exogenously expressed GFP-Mahjong protein, and its degradation products, respectively. Note that MDCK cells mainly express the longer Mahjong isoform and that the GFP-tagged shorter isoform of human being Mahjong is definitely exogenously indicated. This result consequently suggests that the shorter Mahjong isoform can substitute for the longer isoform in cell competition. (B) Effect.