Journal: Biomedicines

Article Title: Unmasking Protein Phosphatase 2A Regulatory Subunit B as a Crucial Factor in the Progression of Dilated Cardiomyopathy.

doi: 10.3390/biomedicines12081887

Figure Lengend Snippet: Figure 4. Knockdown of Ppp2r5d impaired the activity of the mitochondrial respiratory chain complex in MCMs. (A) Relative mRNA expression of the mitochondrial genes in each group. (B) Protein expression of mitochondrial complexes assessed by Western blot in each group. Experiments were repeated three independent times. (C) The relative band intensity of NDUFB8, SDHB, UQCRC2, MTCO1, ATP5a, and B56δ were analyzed by Image J software (version 1.4.3.67) and normalized to total VDAC1. (D) Mitochondria were isolated from MCMs, and complex I activity was measured. The enzymatic activity is presented as milliunits (nmol/min) per milligram (mg) of mitochondrial protein. Data are presented as the mean ± SD with p values indicated.

Article Snippet: The following primary antibodies were used: anti-Phospho-STAT3 (Y705) (9145, CST, New York, NY, USA); antiPhospho-STAT3 (S727) (9136, CST, New York, NY, USA); anti-STAT3 (4904; CST, New York, NY, USA); anti-Ppp2r5d (B56δ) (12068-1-AP, Proteintech, Wuhan, China); anti-GADPH-HRP (Pab001H, ESscience, China); anti-VDAC1/2 (10866-1-AP, Proteintech, Wuhan, China); and anti-Total OXPHOS Rodent WB antibody Cocktail (ab110413, Abcam, Cambridge, UK).

Techniques: Knockdown, Activity Assay, Expressing, Western Blot, Software, Isolation

Journal: Molecular Biology of the Cell

Article Title: APC FZR1 prevents nondisjunction in mouse oocytes by controlling meiotic spindle assembly timing

doi: 10.1091/mbc.E12-05-0352

Figure Lengend Snippet: Premature spindle elongation is independent of CDK1 activity or TPX2, HURP, and Eg5 protein level in Fzr1 −/− oocytes. (A) CDK1 activity measured using an enzyme-linked immunosorbent assay in Fzr1 −/− and control fl/fl oocytes at 2.5 h after GVB. Activity levels were significantly higher in Fzr1 −/− oocytes (* p = 0.037, one-way ANOVA with Tukey's test). Treatment with 2.5 μM flavopiridol reduced CDK1 activity to levels that were not significantly different from control oocytes. (B) Spindle length for control and Fzr1 −/− oocytes, with and without flavopiridol treatment, measured 2.5 h after GVB. Flavopiridol treatment of Fzr1 −/− oocytes did not significantly alter spindle length. (C) Representative immunoblots for TPX2, HURP, and Eg5 on protein from GV-arrested oocytes collected from Fzr1 −/− mice or controls, with n = 50–100 oocytes per lane and at least two separate experiments performed. (D) Densitometric analysis of immunoblots in C. Error bars show SD.

Article Snippet: Immunoblotting was performed using antibodies for FZR1 (ab3242; Abcam, Cambridge, United Kingdom), gylceraldehyde-3-phosphate dehydrogenase (G9545; Sigma-Aldrich), Aurora A kinase (ab13824; Abcam), cyclin B1 (ab72; Abcam), securin (ab3305; Abcam), CDC20 (sc8358; Santa Cruz Biotechnology, Santa Cruz, CA), TPX2 (ab32795; Abcam), Hurp (sc98809; Santa Cruz Biotechnology), Eg5 (NB100-8467; Novus Biologicals, Littleton, CO), γ-tubulin (T6557; Sigma-Aldrich), and secondary immunoglobulin horseradish peroxidase (Dako, Glostrup, Denmark).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Control, Western Blot