Hela dh cells

Hela DH cells (Sigma, cat. 96112022) were grown in DMEM (Gibco, catalog no. 41965-039) supplemented with 10% FBS (Gibco, catalog no. 10106169), plated on glass coverslips. Using Lipofectamine 3000, cells were transfected with either wild-type or mutated connexin constructs in order to overexpress, respectively, Cx26WT or Cx26C169Y proteins. Twenty-four hours after transfection, cells were fixed in 2% paraformaldehyde, saturated and permeabilized for 30 min in BSA 2% Tween 0.1% and incubated over night with a primary antibody against Cx26 (2.5 µg/ml, Connexin 26 Polyclonal Antibody, Rabbit, Invitrogen Catalog Number 71-0500) dissolved in BSA 1% (rinse solution). After three washes in rinse solution, cells were incubated with a secondary antibody [5 µg/ml, Alexa Fluor® 488 Goat Anti-Rabbit IgG (H + L), Invitrogen, Catalog Number A-11008]. After three further washes in rinse solution, glass coverslips with adherent cells were mounted with Fluor Save™ Reagent (Calbiochem, Cat # 345789) on a glass microscope slide. Cell preparations were analyzed using a confocal microscope (TCS SP5, Leica) equipped with an oil-immersion objective (63 × HCX PL APO 1.4 N.A., Leica). Laser line intensities and detector gains were carefully adjusted to minimize signal bleed through outside the designated spectral windows.