mouse polyclonal antibody against gapdh  (Santa Cruz Biotechnology)

Journal: Cells

Article Title: Ubiquitin-Specific Protease 1 Promotes Bladder Cancer Progression by Stabilizing c-MYC

doi: 10.3390/cells13211798

Figure Lengend Snippet: USP1 overexpression promotes bladder cancer cell proliferation, migration, and invasion. ( A ) T24 cells were transfected with FLAG-USP1 or FLAG-USP1 C90S, as indicated, and protein levels were measured by Western blotting. GAPDH served as a control. ( B ) CCK-8 assays were used to analyze cell proliferation ( n = 6). ( C ) Colony formation assays were performed to evaluate cell viability. The colonies were stained with crystal violet and photographed. The number of colonies was determined and plotted ( n = 3). ( D ) Transwell experiments were used to evaluate the effects of USP1 overexpression on cell migration. The cells were imaged (left, ×20 magnification) and counted, and the results were plotted (right, n = 3). The data (mean ± SEM) are representative of 3 independent experiments. ( E ) Transwell chambers with Matrigel were used to evaluate the effects of USP1 overexpression on cell invasion. The cells were imaged (left, ×20 magnification) and counted, and the results were plotted (right, n = 3). The data (mean ± SEM) are representative of 3 independent experiments. Statistical significance was analyzed by ANOVA or Student’s t test. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: Rabbit polyclonal antibody (pAb) against USP1 (14346-1-AP) and mouse mAb against GAPDH (60004-1) were purchased from Proteintech (Wuhan, China).

Techniques: Over Expression, Migration, Transfection, Western Blot, Control, CCK-8 Assay, Staining

Journal: Cells

Article Title: Ubiquitin-Specific Protease 1 Promotes Bladder Cancer Progression by Stabilizing c-MYC

doi: 10.3390/cells13211798

Figure Lengend Snippet: USP1 deficiency inhibits cell proliferation, migration, and invasion. ( A ) USP1 protein levels in WT and USP1-deficient UMUC3 cells were measured by Western blotting, with GAPDH as a loading control. ( B ) Colony formation assays showed the viability of USP1-deficient UMUC3 bladder cancer cells. Colonies were stained with crystal violet and subsequently imaged (left). The number of colonies was determined and plotted (right, n = 3). ( C ) Cell proliferation was analyzed by CCK-8 assays with daily measurements for 5 days ( n = 6). ( D ) Transwell experiments were used to evaluate the effects of USP1 deficiency on UMUC3 bladder cancer cell migration. The cells were imaged (left, ×20 magnification) and counted, and the results were plotted (right, n = 3). ( E ) Transwell chambers with Matrigel were used to evaluate the effects of USP1 deficiency on UMUC3 bladder cancer cell invasion. The cells were imaged (left, ×20 magnification) and counted, and the results were plotted (right, n = 3). The data (mean ± SD) are representative of 3 independent experiments. Statistical significance was analyzed by ANOVA or Student’s t test. *** p < 0.001.

Article Snippet: Rabbit polyclonal antibody (pAb) against USP1 (14346-1-AP) and mouse mAb against GAPDH (60004-1) were purchased from Proteintech (Wuhan, China).

Techniques: Migration, Western Blot, Control, Staining, CCK-8 Assay

Journal: Cells

Article Title: Ubiquitin-Specific Protease 1 Promotes Bladder Cancer Progression by Stabilizing c-MYC

doi: 10.3390/cells13211798

Figure Lengend Snippet: USP1 interacts with c-MYC and promotes c-MYC protein stability. ( A ) Immunofluorescence images of HA-USP1 (red) and FLAG-c-MYC (green) in HEK293T cells. DAPI was used as a nuclear stain (blue). ( B , C ) Immunoprecipitation experiments showed the interaction between USP1 and c-MYC in HEK293 cells. ( D ) Western blot analysis of c-MYC expression in USP1-overexpression T24 cells. ( E ) Western blot analysis of c-MYC expression in USP1-deficient UMUC3 cells. ( F ) Cells were transfected with increasing amounts of the HA-USP1 (0, 200, 400, and 800 ng), HA-USP1 C90S (400 and 800 ng), and FLAG-c-MYC plasmids, and Western blotting was performed to determine the effect of USP1 protein levels on c-MYC expression in HEK293T cells. ( G ) Cells were transfected with FLAG-c-MYC with HA-USP1 or HA-USP1 C90S, as indicated. Western blot analysis of c-MYC stability after treatment with CHX (50 μg/mL) for the indicated time. GAPDH served as a control. ( H ) Cells were co-transfected with FLAG-c-MYC and Myc-Ub with or without HA-USP1 or HA-USP1 C90S, treated with MG132 (10 μM) for 6 h, and then subjected to ubiquitination assays.

Article Snippet: Rabbit polyclonal antibody (pAb) against USP1 (14346-1-AP) and mouse mAb against GAPDH (60004-1) were purchased from Proteintech (Wuhan, China).

Techniques: Immunofluorescence, Staining, Immunoprecipitation, Western Blot, Expressing, Over Expression, Transfection, Control, Ubiquitin Proteomics

Journal: Development (Cambridge, England)

Article Title: A lissencephaly-associated BAIAP2 variant causes defects in neuronal migration during brain development.

doi: 10.1242/dev.201912

Figure Lengend Snippet: Fig. 7. The p.Arg29Trp variant interferes with BAIAP2 localization to the cell membrane. (A) The cellular localization of WT BAIAP2 and p.Arg29Trp (green) in transfected N2a cells. Cells were stained with phalloidin and DAPI to label actin filaments (red) and nuclei (blue), respectively. WT BAIAP2 was distributed to the cell margin, similar to the distribution of actin filaments. In contrast, the p.Arg29Trp variant dispersed its distribution to the cytoplasm. Scale bar: 10 μm. Intensity plots on the right show BAIAP2 (green) and actin filament (red) signals through the line crossing the cell in the left-hand panels. Green and pink shading represents the s.d. of the intensity among seven different cells. (B) WT BAIAP2 and p.Arg29Trp protein in the cytoplasmic and membrane fractions of transfected N2a cells. GAPDH and pan-cadherin were used as cytoplasmic and membrane markers. The bar graph shows BAIAP2 protein levels in cytoplasmic and membrane fractions relative to WT. The p.Arg29Trp variant is significantly increased in the cytoplasmic fraction and decreased in the membrane fraction. Error bars represent s.e.m. *P<0.05, ***P<0.001 (two-tailed unpaired t-test); n=6.

Article Snippet: Primary antibodies included rabbit polyclonal antibody against BAIAP2 (1:200; GTX115421, GeneTex), chicken polyclonal antibody against GFP (1:7500; ab13970, Abcam), mouse monoclonal antibody against Flag tag (1:2000; 66008-2-Ig, Proteintech), rabbit monoclonal antibody against pancadherin (1:2000; GTX61612, GeneTex) and mouse monoclonal antibody against GAPDH (1:10,000; 60004-1-lg, Proteintech).

Techniques: Variant Assay, Membrane, Transfection, Staining, Two Tailed Test