6/7/2023 0 Comments Gapdh western blotFor Db, interaction P = 0.06, genotype P = 0.06 and age P = 0.005. For Da, interaction P = 0.17, genotype P = 0.7 and age P = 0.23. For Bb, interaction P = 0.83, genotype P = 0.21 and age P = 0.0001. For Ba, interaction P = 0.32, genotype P = 0.38 and age P = 0.005. The two factors were the genotype and the age. ![]() Statistical analysis for the effect of the variant across age was by two-way ANOVA. D, boxplots quantifying the Kv7.3 subunit relative to GAPDH in superficial ( Da ) and deep layers ( Db ) of the motor cortex from KCNQ2 WT/WT (black dots) and KCNQ WT/T274M mice (blue dots and purple dots) aged 7, 21 and 30 days. C, western blots of Kv7.3 and of GAPDH in both superficial ( Ca ) and deep layers ( Cb ). Each dot corresponds to quantification in one mouse. B, boxplots quantifying the Kv7.2 subunit relative to the housekeeping protein GAPDH in superficial ( Ba ) and deep layers ( Bb ) of the motor cortex from KCNQ2 WT/WT (black dots) and KCNQ WT/T274M mice (blue dots and purple dots) aged 7, 21 and 30 days. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).ġ0 Figure Kv7.2 and Kv7.3 subunits expression in superficial and deep layers in the motor cortex of developing KCNQ2 WT/WT and KCNQ2 WT/T274M mice A, western blots of Kv7.2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) at postnatal day (PND) 7, 21 and 30 in superficial layers ( Aa ) and deep layers ( Ab ). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10% Bis-Tris gel (Product # NP0302BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). A 37 kDa band corresponding to GAPDH was observed across the cell lines tested. ![]() The blot was probed with Anti-GAPDH Polyclonal Antibody (Product # PA1-987, 1:1,000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Western blot analysis was performed on whole cell extracts (30 µg lysate) of A-431 (Lane 1), COS-7 (Lane 2), MDCK (Lane 3), PC-3 (Lane 4) and tissue extract (30 µg lysate) of Ms Brain (Lane 5).
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