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Table1 Information of the detected CIC rare missense variants in infants with NTDs
Figure 1 Identification of CIC rare missense variants in infants with NTDs. (A) Protein amino acid locus of CIC missense variants. (B) Amino Acid conservation of identified variants among different species.
Figure 2 Subcellular localization and protein abundance of CIC wildtype and CIC variants. (A) Hela cells were transfected with mutated and wildtype constructs of GFP-tagged CIC and pEGFP backbone vector for 36h and were imaged under deconvolution microscope. Scale bar 5um. (B) Western Blotting was performed in Hela cells 48h after transfection. GAPDH was used as loading control. (C) Western Blotting was repeated for three times and student t-test was performed to compare the protein level between wildtype and mutant.
Figure 3 Overexpression of CIC mutants affected FOLR1 protein level and folate binding ability of Hela cells. (A) Hela cells were transfected with mutated and wildtype constructs of GFP-tagged CIC and pEGFP backbone vector for 48h, and western blotting was performed to quantify FOLR1 protein level in each group. GAPDH was used as loading control. (B) Western blotting was repeated for three times and Student’s t-test was performed to compare the protein level between wildtype and mutant. (C) Folate of Hela cells transfected with CIC wildtype and variants were collected and quantified in triplicates.
Figure 4 Overexpression of CIC mutants affected core PCP protein Vangl2 and its downstream protein RhoA in Hela cells. (A) Hela cells were transfected with mutated and wildtype constructs of GFP-tagged CIC and pEGFP backbone vector for 48h, and western blotting was performed to quantify Vangl2 and RhoA protein level in each group. GAPDH was used as loading control. (B) Western blotting was repeated for three times and Student’s t-test was performed to compare Vangl2 and RhoA protein level between wildtype and mutant.
Figure 5 CIC loss of function diminished core PCP protein Vangl2 and its downstream protein RhoA in NIH3T3 cells. (A) NIH3T3 cells were transfected with CIC wildtype, CIC-R353X construct and pEGFP backbone vector for 48h, and Western Blotting was performed to quantify GFP-CIC, Vangl2 and RhoA protein level in each group. GAPDH was used as loading control. (B) Western Blotting was repeated for three times and student t-test was performed to compare Vangl2 and RhoA protein level between wildtype and R353X.