Journal: BMC Nephrology

Article Title: Messenger RNA levels of podocyte-associated proteins in subjects with different degrees of glucose tolerance with or without nephropathy

doi: 10.1186/1471-2369-14-214

Figure Lengend Snippet: Medians and 25th-75th percentiles (P25-P75) of log10 mRNA of podocyte-associated proteins in urine by stage of diabetic nephropathy, in subjects with prediabetes and in controls

Article Snippet: This mix contains AmpliTaq Gold® DNA Polymerase, AmpErase® UNG, ROX passive reference, buffer, and dNTPs (Applied Bioystems, Foster City, CA, USA), as well as gene-specific primers for the mRNA amplification of the following genes (all from Applied Bioystems, Foster City, CA, USA): NPSH1, nephrin (ID: Hs00190446_m1); NPSH2, podocin (ID: Hs00387817_m1); podocalyxin (ID: Hs01574644_m1); synaptopodin (ID: Hs00326493_m1); alpha-actinin-4 (ID: Hs00245168_m1); TRPC6 (ID: Hs00395102_m1); and TGF-β 1 (ID: Hs00998133_m1).

Techniques:

Journal: BMC Nephrology

Article Title: Messenger RNA levels of podocyte-associated proteins in subjects with different degrees of glucose tolerance with or without nephropathy

doi: 10.1186/1471-2369-14-214

Figure Lengend Snippet: Poisson regression analysis of the independent effect of mRNA urinary levels of podocyte-specific proteins on the outcome (albuminuria ≥30 mg/g creatinine) adjusted for age, gender, renal function and presence of diabetes

Article Snippet: This mix contains AmpliTaq Gold® DNA Polymerase, AmpErase® UNG, ROX passive reference, buffer, and dNTPs (Applied Bioystems, Foster City, CA, USA), as well as gene-specific primers for the mRNA amplification of the following genes (all from Applied Bioystems, Foster City, CA, USA): NPSH1, nephrin (ID: Hs00190446_m1); NPSH2, podocin (ID: Hs00387817_m1); podocalyxin (ID: Hs01574644_m1); synaptopodin (ID: Hs00326493_m1); alpha-actinin-4 (ID: Hs00245168_m1); TRPC6 (ID: Hs00395102_m1); and TGF-β 1 (ID: Hs00998133_m1).

Techniques:

Journal: BMC Nephrology

Article Title: Messenger RNA levels of podocyte-associated proteins in subjects with different degrees of glucose tolerance with or without nephropathy

doi: 10.1186/1471-2369-14-214

Figure Lengend Snippet: Diagnostic performance of nephrin, podocalyxin, TRPC6, and alpha-actinin-4 in the diagnosis of albuminuria >30 mg/g creatinine, as determined by the receiver operating characteristic (ROC) curve with related accuracy parameters, expressed as percentages. AUC: area under the curve; PPV: positive predictive value; NPV: negative predictive value.

Article Snippet: This mix contains AmpliTaq Gold® DNA Polymerase, AmpErase® UNG, ROX passive reference, buffer, and dNTPs (Applied Bioystems, Foster City, CA, USA), as well as gene-specific primers for the mRNA amplification of the following genes (all from Applied Bioystems, Foster City, CA, USA): NPSH1, nephrin (ID: Hs00190446_m1); NPSH2, podocin (ID: Hs00387817_m1); podocalyxin (ID: Hs01574644_m1); synaptopodin (ID: Hs00326493_m1); alpha-actinin-4 (ID: Hs00245168_m1); TRPC6 (ID: Hs00395102_m1); and TGF-β 1 (ID: Hs00998133_m1).

Techniques: Diagnostic Assay, Biomarker Discovery

Journal: The Journal of Biological Chemistry

Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

doi: 10.1074/jbc.M111.238360

Figure Lengend Snippet: Schema of TRPC3/TRPC6 chimera. A, domains and motifs in the TRPC3 C terminus. B, representation of the proximal (C1) and distal (C2) C termini of TRPC3 and TRPC6. C, schematic models of TRPC3 chimeras: TRPC3-C6C1, TRPC3-C6C2, and TRPC3-C6TRP. D, schematic models of TRPC6 chimeras: TRPC6-C3C1, TRPC6-C3C2, TRPC6-C3TRP, and TRPC6-C3TRP-C3C2. For B–D, the origin of the TRP domain is shown by the color of the oval. The amino acid (AA) number at the beginning and at the end of the C1 and C2 domains contributed by TRPC3 (top) or TRPC6 (bottom) is indicated. FLAG-tagged chimeras expressed FLAG at the N terminus, and V5-tagged chimeras expressed V5 at the C terminus of the channel.

Article Snippet: Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

doi: 10.1074/jbc.M111.238360

Figure Lengend Snippet: TRP domains, leucine zipper motifs, and AMPK binding site in TRPC3 and TRPC6. A, amino acid compositions of TRP domains and leucine zipper motifs of TRPC3 and TRPC6 are shown. Letters in boldface type indicate exchanged amino acids in the chimeras. For TRP domain exchange, the boldface sequences in the TRP domain of TRPC6 were exchanged with those of TRPC3 to create TRPC3-C6TRP. The boldface amino acids of TRPC3 TRP were exchanged with those of TRPC6 to create TRPC6-C3TRP. For leucine zipper exchange, the boldface sequences in the leucine zipper of TRPC6 were exchanged with those of TRPC3 to create TRPC3-C6LZ. The boldface amino acids of TRPC3 were exchanged with those of TRPC6 leucine zipper to create TRPC6-C3LZ. B, exchange of TRPC3 741–748 and TRPC6 802–809 is shown. Amino acids contributed by TRPC3 (top) or TRPC6 (bottom) are indicated, and localization to the C1 or C2 part of the C terminus is shown.

Article Snippet: Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

doi: 10.1074/jbc.M111.238360

Figure Lengend Snippet: Role of TRP domains in regulation of TRPC3 and TRPC6 by Epo-R. HEK 293T cells were transfected with BFP-TRPC3, BFP-TRPC6, BFP-TRPC3-C6TRP, or BFP-TRPC6-C3TRP chimeras and Epo-R. Fura Red-loaded cells were treated with 40 units/ml Epo. To quantitate [Ca2+]i, F440/F490 was measured at base line and by monitoring over 20 min after Epo stimulation. Shown is the percentage increase in F440/F490 above base line (mean ± S.E. (error bars) percentage increase) = peak F440/F490 divided by base line F440/F490 × 100% − 100% (base line). The numbers of individual cells studied were as follows: BFP-TRPC3 (PBS 17, Epo 21), BFP-TRPC6 (PBS 17, Epo 22), BFP-TRPC3-C6TRP (PBS 16, Epo 22), or BFP-TRPC6-C3TRP (PBS 18, Epo 21). The Epo-stimulated increase in cells expressing TRPC6 and Epo-R is not statistically different from cells expressing Epo-R alone and is thought to be secondary to Epo-R activation of low levels of endogenous channels (17). *, significantly greater percentage increase in F440/F490 compared with Epo-stimulated cells expressing wild type TRPC6 (p < 0.001). **, significantly less percentage increase in F440/F490 compared with Epo-stimulated cells expressing wild type TRPC3 (p < 0.001).

Article Snippet: Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody.

Techniques: Transfection, Expressing, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

doi: 10.1074/jbc.M111.238360

Figure Lengend Snippet: Role of the distal C terminus of TRPC3 and TRPC6 in regulation by Epo-R. HEK 293T cells were transfected with BFP-TRPC3, BFP-TRPC6, BFP-TRPC6-C3TRP, BFP-TRPC6-C3C2, BFP-TRPC6-C3TRP-C3C2, BFP-TRPC3-C6 802–809, or BFP-TRPC6-C3TRP-C3 741–748 chimeras and Epo-R. Fura Red-loaded cells were treated with 40 units/ml Epo. To quantitate [Ca2+]i, F440/F490 was measured at base line and by monitoring over 20 min after Epo stimulation. Shown is the percentage increase in F440/F490 above base line (mean ± S.E. (error bars) percentage increase) = peak F440/F490 divided by base line F440/F490 × 100% − 100% (base line). The numbers of individual cells studied were as follows: BFP-TRPC3 (PBS 56, Epo 102), BFP-TRPC6 (PBS 57, Epo 103), BFP-TRPC6-C3TRP (PBS 20, Epo 34), BFP-TRPC6-C3C2 (PBS 20, Epo 35), BFP-TRPC6-C3TRP-C3C2 (PBS 30, Epo 64), BFP-TRPC3-C6 802–809 (PBS 26, Epo 49), or BFP-TRPC6-C3TRP-C3 741–748 (PBS 12, Epo 30). *, significantly greater percentage increase in F440/F490 compared with Epo-stimulated cells expressing wild type TRPC6 (p < 0.001). **, significantly less percentage increase in F440/F490 compared with Epo-stimulated cells expressing wild type TRPC3 (p < 0.001).

Article Snippet: Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody.

Techniques: Transfection, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

doi: 10.1074/jbc.M111.238360

Figure Lengend Snippet: Plasma membrane insertion of TRPC3/TRPC6 chimeras detected with cell surface biotinylation. HEK 293T cells transfected (Tx'd) with Epo-R and V5-TRPC3, V5-TRPC3-C6TRP, V5-TRPC6, V5-TRPC6-C3TRP, V5-TRPC6-C3C2, V5-TRPC6-C3TRP-C3C2, or V5-TRPC6-C3TRP-C3 741–748 were stimulated with 0–40 units/ml Epo for 0–5 min. Biotinylation of cell surface proteins was performed, and V5-tagged proteins were immunoprecipitated (IP) from lysates with anti-V5 antibody. Western blots (WB) of immunoprecipitates were probed with streptavidin-HRP to detect biotinylated protein and then stripped and reprobed with anti-V5-HRP to detect total protein. Representative results of Western blots from three experiments are shown. Biotinylated and total protein bands were quantitated with densitometry, and the ratio was normalized to time 0. The mean ± S.E. (error bars) values of the biotinylated/total protein ratios from three experiments after 5 min of stimulation are shown. *, significant difference in the ratio compared with time 0 (p < 0.05).

Article Snippet: Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody.

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

doi: 10.1074/jbc.M111.238360

Figure Lengend Snippet: Subcellular localization of TRPC3, TRPC6, TRPC3/TRPC6 chimeras, PLCγ, and Epo-R. A, proteins from HEK 293T cells transfected with FLAG-TRPC3, FLAG-TRPC3-C6TRP, FLAG-TRPC6, FLAG-TRPC6-C3TRP-C3C2, and Epo-R were fractionated, purified, and analyzed by Western blotting (WB). Transfected FLAG-tagged constructs were detected by probing with anti-FLAG antibody. Transfected Epo-R was detected with anti-Epo-R antibody, and endogenous PLCγ was detected with anti-PLCγ antibody. Results from four fractionation experiments using FLAG-tagged constructs and two experiments using V5-tagged constructs were similar, and representative results with FLAG constructs are shown. B, representative results showing quality of fractionation by probing Western blots with anti-GAPDH, anti-Na+K+-ATPase, anti-lamin, and anti-vimentin (markers for cytosol, membrane, nuclear, and cytoskeletal fractions, respectively).

Article Snippet: Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody.

Techniques: Transfection, Purification, Western Blot, Construct, Fractionation

Journal: The Journal of Biological Chemistry

Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

doi: 10.1074/jbc.M111.238360

Figure Lengend Snippet: Membrane and cytoskeletal association of TRPC3, TRPC6, and TRPC3/TRPC6 chimeras. Proteins from transfected HEK 293T cells (A–C) or UT-7/Epo cells (C) were fractionated, purified, and analyzed by Western blotting (WB). Transfected FLAG-tagged constructs were detected by probing with anti-FLAG antibody. Quality of fractionation was confirmed by probing Western blots with anti-Na+K+-ATPase (membrane), anti-vimentin (cytoskeletal fraction), and anti-GAPDH (cytosol marker) antibodies. Equivalent input from lysates was confirmed by probing with anti-actin. A, fractionation with the Qiagen cell fractionation kit. Membrane and cytoskeletal association of TRPC3/6 channels and chimeras. Three to nine experiments (depending on the FLAG-tagged construct) were performed, and representative results are shown. B, fractionation using the 0.5% Triton X-100 extraction method. Representative results of three experiments are shown. C, subcellular fractionation of endogenous TRPC3 and TRPC6 in UT-7/Epo cells with the Qiagen fractionation kit. For all preparations, 100 μg/lane was loaded except for the cytoskeletal fraction of transfected cells, where 50 μg was loaded per lane. Endogenous TRPC3 was detected using anti-TRPC3-C antibody. Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody. Transfected HEK 293T cells were used as controls. Representative results of four experiments are shown. lys, whole cell lysate; M, membrane fraction; Csk, cytoskeletal fraction. *, bands that did not disappear with peptide blocking, indicating that they are nonspecific.

Article Snippet: Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody.

Techniques: Transfection, Purification, Western Blot, Construct, Fractionation, Marker, Cell Fractionation, Blocking Assay

Journal: Kidney international

Article Title: Fibroblast growth factor 23 and fibroblast growth factor receptor 4 promote cardiac metabolic remodeling in chronic kidney disease

doi: 10.1016/j.kint.2025.01.024

Figure Lengend Snippet: ( a ) Treatment of neonatal rat ventricular myocyte cardiobundles with fibroblast growth factor (FGF) 23 for 20 minutes significantly increased contractile force, whereas 7 days of chronic treatment led to a significant reduction in contractile force that could be rescued by coapplication of BLU9931, a selective FGFR4 inhibitor. ( b ) Electrophysiological function was evaluated by pacing of cardiobundles and application of Di-4-ANEPPS (6-[2-(N,N-Dibutylamino)naphthyl]ethenyl-4′-pyridinium propanesulfonate) as voltage-sensitive dye. Chronic exposure of cardiobundles to FGF23 lead to significantly longer action potential durations. ( c ) FGF23-treated bundles exhibited significantly lower conduction velocity that was normalized after coapplication of BLU9931. Besides functional changes, chronic FGF23 treatment also led to cardiobundle hypertrophy, indicated by the ( d,g ) significant increase in cross-section and ( e ) increased expression of hypertrophic mRNA markers Rcan1 and Trpc6 . Increased expression of Rcan1 and Trpc6 was blocked by parallel treatment with BLU9931. ( f ) Metabolic transcription factors that were increased in chronic kidney disease mice also increased in cardiobundles after FGF23 treatment. ( g ) Representative images of cardiobundles indicate cellular hypertrophy after FGF23 treatment by increased myocyte cross-sections. Bars = 10 μm. ( h ) Gene set enrichment analysis of control and FGF23-treated cardiobundles showed an enrichment of metabolic pathways, particularly fatty acid metabolism, adipogenesis, and cholesterol homeostasis. ( i ) Additional enrichment was detected in pathways related to mitochondrial function, such as oxidative phosphorylation, respiratory chain, organelle fission, and organelle inner membrane. Downregulated pathways after FGF23 treatment include angiogenesis, vascular development, tumor necrosis factor (TNF)-α signaling, and P53. Bar graphs represent mean ± SEM with individual values included in the graph. n ≥ 3 for all experiments. * P < 0.05, ** P < 0.005, **** P < 0.0001. APD, action potential duration; DAPI, 4′,6-diamidino-2-phenylindole; ES, enrichment score; FDR, false discovery rate; NES, normalized enrichment score. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org .

Article Snippet: Trpc6 , Mm01176083_m1.

Techniques: Functional Assay, Expressing, Control, Phospho-proteomics, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Collagen receptor cross-talk determines α-smooth muscle actin-dependent collagen gene expression in angiotensin II–stimulated cardiac fibroblasts

doi: 10.1074/jbc.RA119.009744

Figure Lengend Snippet: α-SMA–dependent TRPC6 regulates collagen type I expression in Ang II–treated cardiac fibroblasts. Cardiac fibroblasts were transiently transfected with α-SMA siRNA or scrambled siRNA (control). A and B, following exposure of the transfected cells to Ang II for 12 h, collagen α1(I) protein expression was examined by Western blot analysis and normalized to β-actin. ***, p < 0.001 versus control; ###, p < 0.001 versus Ang II. C and D, following exposure of the transfected cells to Ang II for 12 h, TRPC6 protein expression was examined by Western blot analysis and normalized to β-actin. ***, p < 0.001 versus control; ###, p < 0.001 versus Ang II. E and F, cardiac fibroblasts were transiently transfected with TRPC6 siRNA or scrambled siRNA (control). Following exposure of the transfected cells to Ang II for 12 h, collagen type I protein expression was examined by Western blot analysis and normalized to β-actin. ***, p < 0.001 versus control; ###, p < 0.001 versus Ang II. G and H, cardiac fibroblasts were transiently co-transfected with α-SMA siRNA and TRPC6 plasmid overexpression vector. Following revival of the transfected cells and serum deprivation, cardiac fibroblasts were exposed to Ang II for 12 h. Collagen α1(I) protein expression was examined by Western blot analysis and normalized to β-actin. ***, p < 0.001 versus control; ###, p < 0.001 versus Ang II; †††, p < 0.001 versus Ang II + α-SMA siRNA; ns, not significant versus Ang II. Data are representative of three independent experiments (n = 3). Error bars, S.D.

Article Snippet: The TRPC6 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_053559","term_id":"16758329","term_text":"NM_053559"}} NM_053559 ) rat tagged ORF clone was from Origene (Rockville, MD).

Techniques: Expressing, Transfection, Western Blot, Plasmid Preparation, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: Collagen receptor cross-talk determines α-smooth muscle actin-dependent collagen gene expression in angiotensin II–stimulated cardiac fibroblasts

doi: 10.1074/jbc.RA119.009744

Figure Lengend Snippet: α-SMA–dependent TRPC6 regulates collagen type I expression via YAP activation in Ang II–treated cardiac fibroblasts. Cardiac fibroblasts were transiently transfected with α-SMA siRNA, TRPC6 siRNA, or scrambled siRNA (control). A and B, following exposure of the α-SMA siRNA-transfected cells to Ang II for 6 h, phosphorylation of YAP at Ser-127 was examined by Western blot analysis and normalized to total YAP. ***, p < 0.001 versus control; **, p < 0.01 versus Ang II. C and D, following exposure of the TRPC6 siRNA–transfected cells to Ang II for 6 h, phosphorylation of YAP at Ser-127 was examined by Western blot analysis and normalized to total YAP. ***, p < 0.001 versus control; **, p < 0.01 versus Ang II. E, cardiac fibroblasts were transiently transfected with α-SMA siRNA, TRPC6 siRNA, or scrambled siRNA (control). Following Ang II treatment for 6 h, the ChIP assay was performed as described under “Experimental procedures.” DNA binding of YAP to the collagen α1(I) gene promoter was confirmed using anti-YAP antibody. A nonspecific anti-rabbit IgG was used as negative control. A representative image showing the PCR amplification product is shown. F and G, subconfluent quiescent cultures of cardiac fibroblasts were pretreated with the YAP inhibitor (verteporfin) for 1 h and, subsequently, with Ang II. Protein was isolated at 12 h post-Ang II treatment and subjected to Western blot analysis for detection of collagen α1(I), with β-actin as loading control. ***, p < 0.001 versus control; ###, p < 0.001 versus Ang II. Data are representative of three independent experiments (n = 3). Error bars, S.D.

Article Snippet: The TRPC6 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_053559","term_id":"16758329","term_text":"NM_053559"}} NM_053559 ) rat tagged ORF clone was from Origene (Rockville, MD).

Techniques: Expressing, Activation Assay, Transfection, Western Blot, Binding Assay, Negative Control, Amplification, Isolation

Journal: The Journal of Biological Chemistry

Article Title: Collagen receptor cross-talk determines α-smooth muscle actin-dependent collagen gene expression in angiotensin II–stimulated cardiac fibroblasts

doi: 10.1074/jbc.RA119.009744

Figure Lengend Snippet: TRPC6-dependent Ca2+ in mediating YAP activation and collagen expression in Ang II–treated cardiac fibroblasts. Subconfluent quiescent cultures of cardiac fibroblasts were pretreated with EGTA (1 mm) for 1 h and, subsequently, with Ang II. A–C, phosphorylation status of YAP at Ser-127 was examined by Western blot analysis at 6 h post-Ang II treatment and normalized to total YAP. *, p < 0.05 versus control; ***, p < 0.001 versus Ang II; ###, p < 0.001 versus Ang II. Expression of collagen α1(I) was analyzed at 12 h post-Ang II treatment with β-actin as loading control. ***, p < 0.001 versus control; **, p < 0.01 versus Ang II; ##, p < 0.01 versus Ang II. D–F, cardiac fibroblasts were transiently transfected with TRPC6 overexpression plasmid or control plasmid. Following exposure of the transfected cells to EGTA (1 mm), phosphorylation status of YAP at Ser-127 was examined by Western blot analysis at 6 h post-EGTA treatment and normalized to total YAP. ***, p < 0.001 versus control; *, p < 0.05 versus TRPC6 OE; **, p < 0.01 versus TRPC6 OE. Collagen α1(I) protein expression was examined by Western blot analysis at 12 h post-EGTA treatment and normalized to β-actin. **, p < 0.01 versus control; ††, p < 0.01 versus TRPC6 OE; ###, p < 0.001 versus TRPC6 OE. G–J, cardiac fibroblasts were transiently transfected with TRPC6 overexpression plasmid or control plasmid. Following exposure of the transfected cells to SKF96365 (10 μm), phosphorylation status of YAP at Ser-127 was examined by Western blot analysis at 6 h post-treatment with SKF96365 and normalized to total YAP. *, p < 0.05 versus control; **, p < 0.01 versus TRPC6 OE; #, p < 0.05 versus TRPC6 OE. The control and SKF96365 blot from a separate run have been juxtaposed. Collagen α1(I) protein expression was examined by Western blot analysis at 12 h post-treatment with SKF96365 and normalized to β-actin. ***, p < 0.001 versus control; ###, p < 0.001 versus TRPC6 OE; †††, p < 0.001 versus TRPC6 OE. Data are representative of three independent experiments (n = 3). Error bars, S.D.

Article Snippet: The TRPC6 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_053559","term_id":"16758329","term_text":"NM_053559"}} NM_053559 ) rat tagged ORF clone was from Origene (Rockville, MD).

Techniques: Activation Assay, Expressing, Western Blot, Transfection, Over Expression, Plasmid Preparation