primary polyclonal rabbit anti trpc6 antibody  (Alomone Labs)

Journal: Nucleic Acids Research

Article Title: A novel transient receptor potential C3/C6 selective activator induces the cellular uptake of antisense oligonucleotides

doi: 10.1093/nar/gkae245

Figure Lengend Snippet: Effects of the TRPC3/C6/C7 inhibitor, knockdown of TRPC3/C6, and Ca 2+ chelator on ASO uptake, and examination of L687-mediated uptake pathways. ( A ) ASO uptake was analysed by incubating cells with or without a TRPC inhibitor (SKF96365). Alexa647-AmNA#26 (10 nM) was added to either 10 μM L687, 20 μM GSK1702934A, or 30 μM CBD with or without 20 μM SKF96365 in the medium. After 24 h, the intracellular fluorescence intensities were analysed by flow cytometry. Data are shown as the relative MFI of ASO in DMSO. All data are presented as mean ± standard error of the mean (SEM) of three independent experiments ( n = 3). Statistical significance was determined by comparing with values of DMSO using Tukey's test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B ) siRNA-mediated knockdown of the TRPC3/C6 channels. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed and compared with that in the untreated cells. ( C ) siRNA-mediated knockdown of TRPC3/C6 channel. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed by western blot and compared with that in untreated cells. ( D ) The effects of siRNA-mediated TRPC3/C6 channel knockdown on ASO uptake. TRPC3 siRNA (30 nM), TRPC6 (10 nM), or a combination of both were transfected into A549 cells for 48 h. The medium was replaced with Alexa647-AmNA#26 containing L687, and intracellular fluorescence intensities were analysed after 24 h. Data are shown as the relative MFI of ASO in DMSO. ( E ) Analysis of ASO uptake after incubating cells with a Ca 2+ chelator (BAPTA-AM). ASO and L687 were added to the medium, with or without 10 μM BAPTA-AM. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( F ) Analysis of dextran uptake mediated by L687. Alexa647-labelled dextran (1 and 3 μM) with 10 and 30 μM L687 was added to the medium, and A549 cells were cultured for 24 h. Intracellular fluorescence was analysed by flow cytometry, and the relative MFI was compared with 1 μM of Alexa647-dextran with DMSO. ( G ) Analysis of ASO uptake by incubating the cells with a macropinocytosis inhibitor (Cytochalasin D). L687 (30 μM) was then added to the medium. The following day, cells were then washed twice with PBS and incubated with cytochalasin D in the medium for 1 h. Then, cells were washed twice with PBS and incubated with Alexa647-AmNA#26 (10 nM) and L687 (30 μM). After 4 h, intracellular fluorescence intensities were analysed by flow cytometry. ( H ) Analysis of ASO uptake by incubating cells with a macropinocytosis inhibitor (EIPA). ASO and L687 were added to the medium with or without 100 μM EIPA. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( I ) Fluorescence imaging analysis of ASO incorporated into cells. Alexa647-AmNA#26 (100 nM) and L687 (30 μM) were added to the medium, and staining with Lysotracker-green and Hoechst, fluorescence microscopy imaging, and image analysis were performed after 48 h. ASO, antisense oligonucleotide; CBD, cannabidiol; DMSO, dimethyl sulfoxide; MFI, mean fluorescence intensity; TRPC, transient receptor potential canonical.

Article Snippet: Incubation with primary polyclonal rabbit anti-TRPC3 antibody (#ACC-016; Alomone Labs, Jerusalem, Israel), primary polyclonal rabbit anti-TRPC6 antibody (#ACC-120; Alomone Labs, Jerusalem, Israel) at 1:2000 dilution and mouse monoclonal anti-GAPDH (#AM4300; Thermo Fisher Scientific, MA, USA) at 1:2000 dilution was performed at 4°C overnight.

Techniques: Knockdown, Fluorescence, Flow Cytometry, Transfection, Expressing, Western Blot, Cell Culture, Incubation, Imaging, Staining, Microscopy