Additional file 1 of Stimulated phosphorylation of ERK in mouse kidney mesangial cells is dependent upon expression of Cav3.1
Creators: name: Pandi, Sudha Priya Soundara
name: Shattock, Michael J.
name: Hendry, Bruce M.
name: Sharpe, Claire C.
Publisher: name: figshare
Year: 2022
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Hematology

description: Additional file 1: Supplementary material. Bacterial transformation, plasmidpurification and validation, selection and validationof single-cell clones. SupplementaryFigure S1. Confirmation of TTCC isoform in Mouse mesangial cells andmodulation of its proliferation by TTCC blockers. (a, b, c) RT-PCR for the Ttype calcium isoforms were detected in mouse mesangial cells (MES13). The bandin each lane in the agarose gel represent the PCR product of the T type calciumisoforms Cacna1G, Cacna1H and Cacna1I (PCR product size: CaV3.1-376; CaV3.2-594;CaV3.3-528). mRNA of the CaV3.3 in 72 h treatment with PDGF and TGFβ1 aloneshown faint band. Supplementary FigureS2. Validation of CRISPR-cas9- gRNA for cacna1G and Cacna1H. plenti-CRISPR-cas9-cacna1G a) andplenti-CRISPR-cas9-cacna1H b) vector map expressing gRNA mapping to exon 4 of36 exons containing cacna1G gene and on exon 6 of 35 exons containing cacna1Hgene under the control of U6 promoter, with selection markers ampicillin forprokaryotic and puromycin for mammalian cell selection. a) and b) plasmids wereconfirmed by restriction digestion by MluI restriction enzyme and by sangersequencing using primer designed for U6 promoter. (gRNA sequences:Cacnca1G-TCCGTGTGCTGCGACCGCTC, Cacna1H- ACCTGACGAA GGCGCTGTCC). Gel: Panel A:Lane 1-10KB ladder, Lane 2 –circular plasmid, Lane 3-No restriction enzymecontrol, Lane 4- plasmid digested by Mlu. Panel B: Lane 1 –circular plasmid,Lane 2-No restriction enzyme control, Lane 3- plasmid digested by Mlu, Lane4-10KB ladder. Yellow highlight - gRNA sequence. Supplementary Figure S3. Confirmation of CaV3.1 and CaV3.2knockout at the genomic level. a) PCR amplificationfor CaV3.1 (left panel) and CaV3.2 (right panel) genes inthe six clones each from cacna1G and cacna1h by using primers spanning gRNA region. Cacna1G clone 2 and Cacna1H clone 3harbouring indel mutation shows PCR product shift compare to control. b)Sequencing analysis of cacna1G and cacna1H knockout in the clone 2 of cacna1G,clone 3 of cacna1H. Sequencing resultsshow deletion of the gRNA sequence with the frameshift mutation compare to wildtype (WT) control, confirming the knockouts. Right panel-sequencing forcacna1G; left panel- sequencing for cacna1H. Yellow highlight represents the sequencebefore gRNA in the gene and grey highlight represent the gRNA sequence. Supplementary Figure S4. Confirmation of cacna1G and cacna1H knockout at thepost-translational level. Western blot for CaV3.1(a) protein expression in the single-cell clones of CaV3.1 knockoutshow absence of bands in all six CaV3.1 KO clones compared to wildtype cells and the positive control mouse brain. (b) single-cell clone of CaV3.2KO shows the absence of bands in 5 clones compared to wild type and positivecontrol HEK cell overexpressing CaV3.2. Loading control- actin. Supplementary FigureS5: Figure shows the full-length image ofWestern blot for Figure 2a described in the main text. Supplementary Figure S6: Figure shows the full-length image ofWestern blot for Figure 3a described in the main text. Supplementary Figure S7: Figure shows the full-length image ofWestern blot for Figure 4a and 4c described in the main text. Supplementary Table S1: Primers list, 1.28 MB, PDF.