Colocalization of Kv7.4, Caveolin-1, and Dynein

Colocalization of dynein with Kv7.4 or caveolin-1 and caveolin-1 with Kv7.4 was studied with PLA in HEK293B cells stably expressing Kv7.4 and Kv7.4-Q580A or freshly isolated rat mesenteric artery myocytes using the Duolink in situ (PLA) detection kit 563 (Olink) per the manufacturer’s instructions. Similar to previous studies (Zhong et al., 2010a (link); Brueggemann et al., 2014 (link); Chadha et al., 2014 (link); Jepps et al., 2015 (link); Stott et al., 2016 (link); Barrese et al., 2020 (link)), cells were allowed to adhere to coverslips and fixed in 4% paraformaldehyde in PBS. Cells were permeabilized in PBST, blocked in Duolink blocking solution, and incubated with pairs of primary antibodies. The primary antibodies employed were dynein (ab23905; Abcam), Kv7.4 (ab65797; Abcam), caveolin-1 (1:500 ab17052; Abcam), caveolin-1 (C3237; Sigma-Aldrich), and NCX (R3F1; Swant). Combinations of secondary anti-rabbit or anti-mouse antibodies of PLA PLUS and MINUS probes were used followed by hybridization, ligation, and amplification steps. Colocalization signals (proteins located within 40 nm of each other) were visualized with a standard Zeiss LSM710 upright laser-scanning confocal microscope.

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van der Horst J., Rognant S., Abbott G.W., Ozhathil L.C., Hägglund P., Barrese V., Chuang C.Y., Jespersen T., Davies M.J., Greenwood I.A., Gourdon P., Aalkjær C, & Jepps T.A. (2021). Dynein regulates Kv7.4 channel trafficking from the cell membrane. The Journal of General Physiology, 153(3), e202012760.

Publication 2021

Duolink in situ (PLA) detection kit 563 ab23905 ab65797 ab17052 LSM710 upright

Anti antibodies Antibodies Caveolin 1 Cells Confocal laser scanning microscope Dynein Hybridization Ligation Mesenteric artery Mouse Myocytes Paraformaldehyde Proteins Rabbit

Corresponding Organization : University of Copenhagen

Other organizations : University of California, Irvine, St George's, University of London, University of Naples Federico II, Lund University, Aarhus University

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Variable analysis

independent variables

Kv7.4 and Kv7.4-Q580A expression in HEK293B cells
Freshly isolated rat mesenteric artery myocytes

dependent variables

Colocalization of dynein with Kv7.4 or caveolin-1
Colocalization of caveolin-1 with Kv7.4

control variables

Cell type (HEK293B cells and rat mesenteric artery myocytes)
Fixation method (4% paraformaldehyde in PBS)
Permeabilization (PBST)
Blocking (Duolink blocking solution)
Primary antibodies (dynein, Kv7.4, caveolin-1, NCX)
Secondary antibodies (PLA PLUS and MINUS probes)
Hybridization, ligation, and amplification steps
Visualization (Zeiss LSM710 upright laser-scanning confocal microscope)

controls

Positive control: Previous studies (Zhong et al., 2010a; Brueggemann et al., 2014; Chadha et al., 2014; Jepps et al., 2015; Stott et al., 2016; Barrese et al., 2020) on colocalization of dynein with Kv7.4 or caveolin-1, and caveolin-1 with Kv7.4
Negative control: Not explicitly mentioned

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