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: PLoS Genetics

Article Title: A homozygous missense variant in CACNB4 encoding the auxiliary calcium channel beta4 subunit causes a severe neurodevelopmental disorder and impairs channel and non-channel functions

doi: 10.1371/journal.pgen.1008625

Figure Lengend Snippet: (A and B) HEK293T cells were transfected with empty vector (EV), β 1 -V5, β 4b -V5 or β 4b -L125P-V5 expression construct as indicated. Endogenous PPP2R5D and TNIK were immunoprecipitated from cell extracts using an anti-PPP2R5D (A) and an anti-TNIK antibody (B), respectively, both coupled to magnetic protein G Dynabeads. For IgG control the cell lysate from cells transfected with the β 4b -L125P-V5 mutant construct was incubated with an anti-normal rabbit IgG antibody coupled to Dynabeads. Co-precipitated β 1 -V5 and β 4b -V5 proteins were detected by immunoblotting using anti-V5-HRP and anti-β 4 antibody. A representative blot of four (A) or three (B) independent experiments each is shown.

Article Snippet: For immunoblotting and immunoprecipitation: mouse anti-CACNA2D1 (α 2 δ-1) (20A) antibody (1:500, MA3-921, Thermo Fisher Scientific), mouse anti-Ca V β 4 calcium channel antibody (1:500; 75–054, NeuroMab), mouse anti-GFP antibody (1:5000; 902601, BioLegend), anti-normal mouse IgG (1:100; 12–371, Merck Millipore) and anti-normal rabbit IgG antibody (1:100; 12–370, Merck Millipore), rabbit anti-PPP2R5D antibody (IP 1:25; WB 1:2,500; A301-098A, Bethyl Laboratories Inc.), rabbit anti-TNIK antibody (IP 1:50; WB 1:1,000; 32712, Cell Signaling Technologies), mouse anti-V5 (1:125; R960-25, Thermo Fisher Scientific), mouse anti-V5 tag horseradish peroxidase (HRP)-coupled antibody (1:5,000; R961-25, Thermo Fisher Scientific).

Techniques: Transfection, Plasmid Preparation, Expressing, Construct, Immunoprecipitation, Control, Mutagenesis, Incubation, Western Blot

Journal: PLoS Genetics

Article Title: A homozygous missense variant in CACNB4 encoding the auxiliary calcium channel beta4 subunit causes a severe neurodevelopmental disorder and impairs channel and non-channel functions

doi: 10.1371/journal.pgen.1008625

Figure Lengend Snippet: HEK293T cells were plated on collagen-coated glass slides and transiently transfected with the indicated constructs. β 4b -V5 and β 4b -L125P-V5 were stained by mouse anti-V5 antibody (green); endogenous PPP2R5D was visualized using rabbit anti-PPP2R5D antibody (red). Representative images of two independent experiments are shown. White boxes indicate magnified areas of specimen shown on the very right-hand side. Scale bars, 10 μm. EV: empty vector (control).

Article Snippet: For immunoblotting and immunoprecipitation: mouse anti-CACNA2D1 (α 2 δ-1) (20A) antibody (1:500, MA3-921, Thermo Fisher Scientific), mouse anti-Ca V β 4 calcium channel antibody (1:500; 75–054, NeuroMab), mouse anti-GFP antibody (1:5000; 902601, BioLegend), anti-normal mouse IgG (1:100; 12–371, Merck Millipore) and anti-normal rabbit IgG antibody (1:100; 12–370, Merck Millipore), rabbit anti-PPP2R5D antibody (IP 1:25; WB 1:2,500; A301-098A, Bethyl Laboratories Inc.), rabbit anti-TNIK antibody (IP 1:50; WB 1:1,000; 32712, Cell Signaling Technologies), mouse anti-V5 (1:125; R960-25, Thermo Fisher Scientific), mouse anti-V5 tag horseradish peroxidase (HRP)-coupled antibody (1:5,000; R961-25, Thermo Fisher Scientific).

Techniques: Transfection, Construct, Staining, Plasmid Preparation, Control