antibody anti ddk rabbit polyclonal antibody  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Missense variants in the myosin binding domains of MYBPC3 and MYBPHL impair sarcomere incorporation

doi: 10.1101/2025.09.29.679322

Figure Lengend Snippet: (A) Domain schematic showing full-length cMyBP-C, full-length MyBP-HL, mini-C construct and MyBP-HL constructs. The mini-C construct consists of the three C-terminal domains found in full-length cMyBP-C, critical for myosin binding, and a Myc-DDK tag on the N-terminus. The MyBP-HL construct consists of the full-length MyBP-HL with a Myc-DDK tag on the N-terminus. (B) MyBP-HL construct transfected in neonatal rat ventricular cardiomyocytes and proper sarcomere localization was determined via immunofluorescence staining for endogenous cMyBP-C and MyBP-HL. Co-localization was quantified via Pearson’s R value (N = 2; One-way ANOVA). (C) Mini-C construct transfected in NRVMs and stained using Anti-DDK. Proper sarcomere localization was determined and quantified as previously described in (B). (N = 4; One-way ANOVA).

Article Snippet: Primary antibodies against human cMyBP-C (Santa Cruz, 137180), MyBP-HL (Barefield, 2022; 1:2500) and Anti-DDK (Origene, TA150078), phosphorylated serine 273 cMyBP-C [90], actinin (ProteinTech, 14221-1-AP) were incubated in blocking buffer overnight at room temperature.

Techniques: Construct, Binding Assay, Transfection, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Missense variants in the myosin binding domains of MYBPC3 and MYBPHL impair sarcomere incorporation

doi: 10.1101/2025.09.29.679322

Figure Lengend Snippet: (A) Schematic showing presence of selected missense variants within MyBP-HL construct, all located in the HL3 domain. (B) NRVMs were transfected with the missense variants expressed within MyBP-HL plasmid. Immunofluorescence microscopy was performed to stain for endogenous cMyBP-C (E7), and MyBP-HL. (C) Co-localization was measured by Pearson’s R value, using Image J Co-Loc2 plug-in. N = 4; One-way ANOVA, Tukey’s multiple comparison test. (D) Expression of wild-type MyBP-HL construct and missense variants within MyBP-HL construct within myofilament prepped and supernatant samples of NRVMs was measured via immunoblotting, blotting for its DDK tag. N = 3. (E) Densitometric analysis was quantified for (D).

Article Snippet: Primary antibodies against human cMyBP-C (Santa Cruz, 137180), MyBP-HL (Barefield, 2022; 1:2500) and Anti-DDK (Origene, TA150078), phosphorylated serine 273 cMyBP-C [90], actinin (ProteinTech, 14221-1-AP) were incubated in blocking buffer overnight at room temperature.

Techniques: Construct, Transfection, Plasmid Preparation, Immunofluorescence, Microscopy, Staining, Comparison, Expressing, Western Blot

Journal: bioRxiv

Article Title: Missense variants in the myosin binding domains of MYBPC3 and MYBPHL impair sarcomere incorporation

doi: 10.1101/2025.09.29.679322

Figure Lengend Snippet: (A) Domain schematic showing T2A-P2A gene and its translation mechanism of producing three separate proteins, mini-C with its N-terminal DDK tag, a fluorescent reporter protein, td-tomato, and MyBP-HL. (B) Immunoblot of NRVMs expressing RFP, MyBP-HL, mini-C, and T2A/P2A constructs, showing expression of mini-C and MyBP-HL in positive controls and T2A/P2A construct (N = 3; One-way ANOVA). (C) NRVMs transfected with T2A/P2A construct, showing proper sarcomere localization of mini-C and MyBP-HL using endogenous cMyBP-C. (D) Abundances of mini-C and MyBP-HL normalized to myosin 6 and 7 in NRAMs and NRVMs transfected with T2A/P2A construct (N = 3). (E) MYBPC3 peptide standard curve, measuring abundances of cMyBP-C within positive and negative controls, showing abundances are within range of peptide standard curve.

Article Snippet: Primary antibodies against human cMyBP-C (Santa Cruz, 137180), MyBP-HL (Barefield, 2022; 1:2500) and Anti-DDK (Origene, TA150078), phosphorylated serine 273 cMyBP-C [90], actinin (ProteinTech, 14221-1-AP) were incubated in blocking buffer overnight at room temperature.

Techniques: Western Blot, Expressing, Construct, Transfection

Journal: bioRxiv

Article Title: Missense variants in the myosin binding domains of MYBPC3 and MYBPHL impair sarcomere incorporation

doi: 10.1101/2025.09.29.679322

Figure Lengend Snippet: (A) Schematic of T2A/P2A construct expressing the MYBPC3 missense variants. (B) Representative immunoblots with myofilament and supernatant protein lysates from NRVMs transfected with the wild-type T2A/P2A construct and T2A/P2A with MYBPC3 missense variants, blotting for mini-C, MyBP-HL, and 2A-tdTomato. (C) The ratios of DDK and MyBP-HL were normalized to their 2A expression patterns. A stoichiometry ratio between DDK/2A and MyBP-HL/2A was quantified. N = 6 – 7; One-way ANOVA, Tukey’s multiple comparison test.

Article Snippet: Primary antibodies against human cMyBP-C (Santa Cruz, 137180), MyBP-HL (Barefield, 2022; 1:2500) and Anti-DDK (Origene, TA150078), phosphorylated serine 273 cMyBP-C [90], actinin (ProteinTech, 14221-1-AP) were incubated in blocking buffer overnight at room temperature.

Techniques: Construct, Expressing, Western Blot, Transfection, Comparison

Journal: bioRxiv

Article Title: Missense variants in the myosin binding domains of MYBPC3 and MYBPHL impair sarcomere incorporation

doi: 10.1101/2025.09.29.679322

Figure Lengend Snippet: (A) Schematic of T2A/P2A construct expressing the MYBPHL missense variants. (B) Representative immunoblots with myofilament protein from NRVMs transfected with the wild-type T2A/P2A construct and MYBPHL missense variant expressing T2A/P2A, blotting for mini-C, MyBP-HL, and the 2A peptide fragment on the tdTomato protein. (C) The ratios of DDK and MyBP-HL were normalized to the 2A protein levels. The ratio between MyBP-HL/2A and DDK/2A was quantified. N = 7 – 8; One-way ANOVA, Tukey’s multiple comparison test.

Article Snippet: Primary antibodies against human cMyBP-C (Santa Cruz, 137180), MyBP-HL (Barefield, 2022; 1:2500) and Anti-DDK (Origene, TA150078), phosphorylated serine 273 cMyBP-C [90], actinin (ProteinTech, 14221-1-AP) were incubated in blocking buffer overnight at room temperature.

Techniques: Construct, Expressing, Western Blot, Transfection, Variant Assay, Comparison