Fitc conjugated goat anti rabbit immunoglobulin

Metaphase chromosome spreads were prepared and immunostained as previously described^{32 (link)}. Briefly, cells were treated for two hours with colcemid at 0.1 µg/ml. Cells were washed twice with cold PBS and swollen in 75 mM KCl at 2 × 10⁵ cells/ml at room temperature for 10 min. 200 µl aliquots of the swollen cell suspension were spun onto glass slides at 1800 rpm for 10 min in a Shandon Cytospin 4. Slides were immersed for 10 min. at room temperature in KCM buffer (120 mM KCl, 20 mM NaCl, 10 mM Tris/HCl pH 8.0, 0.5 mM EDTA, 0.1% Triton X-100). Immunolabelling was carried out for 1 h at 4 °C with antisera diluted 200–400 fold in KCM supplemented with 1–1.5%. BSA. The secondary antibody was FITC-conjugated goat anti-rabbit immunoglobulin (Sigma F1262) diluted 150-fold in KCM, 1% BSA. Slides were washed twice in KCM (5 min. at room temperature), fixed in 4% v/v formaldehyde (10 min. at room temperature), rinsed in deionised water and mounted in Vector Shield (Vector lab) supplemented with DAPI (Sigma) at 2 µg/ml. Slides were visualized on a Zeiss Axioplan 2 epifluorescence microscope.

Antibodies were purchased from the following companies: two symplekin monoclonal antibodies (mAbs) were from Progen Biotechnik (cat #651100; Heidelberg, Germany) and BD Biosciences (cat #610644; Rockville, MD, USA); ZO-1 mAb (HPA001637), CSDA mAb (SAB1404593), GAPDH mAb (G8795), rabbit anti-FLAG (F7425) antibody, FITC-conjugated goat anti-rabbit Immunoglobulin G (IgG) antibody (F6005), anti-flag affinity gel (F2426), and 3 × FLAG Peptide (F4799) were from Sigma-Aldrich (St. Louis, MO, USA); ERK1/2 mAb (13–6200) and rabbit anti-β-catenin (AHO0462) antibody were from Invitrogen (Carlsbad, CA, USA); biotinylated rabbit anti-phosphoserine (ab9335) and rabbit anti-phosphothreonine (ab9340) antibodies were from Abcam (Cambridge, MA, USA); rabbit anti-E-cadherin (3195S) antibody was from Cell Signaling Technology (Beverly, MA, USA); tubulin-α mAb (66031) and LMNB1 mAb (66095) were from Proteintech (Wuhan, China); Cy3-conjugated goat anti-mouse IgG antibody (115-165-166) was from Jackson ImmunoResearch Laboratories (West Grove, PA, USA); peroxidase-conjugated goat anti-mouse IgG antibody (HD003-1) was from DingGuo (Beijing, China); and peroxidase-conjugated goat anti-rabbit IgG antibody (M21002) was from Abmart (Shanghai, China).

Cells or mortalin knockdown cells were placed on cover slips in 24-well plates and pretreated with SalB for 2 h prior to exposure to 400 μM H₂O₂ for 2 h. After washing with PBS, the slides were fixed in 4% paraformaldehyde for 10 min at room temperature, washed thrice with PBS, permeabilized with 0.1% saponin, blocked with 10% normal goat serum and incubated overnight at 4 °C with cytochrome c antibody. The slides were incubated with FITC-conjugated goat anti-rabbit immunoglobulin (Sigma-Aldrich, Burlingame, CA, USA) for 2 h. Nuclei were stained with Hoechst 33258 (Beyotime, Jiangsu, China). Cover slips were observed under a fluorescence microscope (Leica Microsystems, Wetzlar, Germany) equipped with a Leica DFC420 camera.