Anti dynll1

The following antibodies were used. Anti-β-Galactosidase (Bioss, bs-4960R), anti-alkaline phosphatase (Novus Biologicals, NBP1-32948); anti-heterochromatin protein 1-γ (HP1-γ, phospho Ser 93, Bioss, bs-3221R); anti-p21 (Bioss, bs-10129R); anti-p27 (phospho Thr 187, Abcam, ab75908); anti-NOS-1, 2 (Thermo Fisher Scientific, PA1-033 and -036); anti-NOS-3 (Sigma-Aldrich, SAB-4300435); anti-ErbB2 (Thermo Fisher Scientific, PA5-16395); anti-pSMAD3 (Ser423/Ser425, Novous Biological, NBP1-77836); anti-CD24 (Novus Biologicals, NBP1-4639055); anti-CD44 (Bioss, bs-2507R); anti-S-Nitroso-Cysteine (Abcam, ab50185 or Alpha Diagnostics, NISC11-A); anti-Integrin α6 (BD Biosciences, 555734); anti-GM130 (Cell Signaling, 12480 S); anti-human CK 14 (ThermoFisher, MA511599); anti-human CK 18 (ThermoFisher, PA514263); anti-mouse CK 14 (BioLegend, 905301); anti-human CK 8/18 (DSHB, Troma-I); anti-Cleaved Caspase3 (Cell Signaling, #9664); anti-β-Actin (Sigma, A1978); anti-DYNLL1 (Abcam, ab51603); and anti-ADMA (EMD Millipore, 09-814).

A Duolink PLA was performed as described previously (Sigma) (17 (link)). Briefly, 1 × 10⁴ cells were plated on a glass slide, fixed, and blocked using the Duolink blocking solution (Sigma). The cells were coincubated overnight with anti-Dynll1 (Abcam) and mouse-Cox4i1 (CST) antibodies at 4°C. The secondary antibodies of the Duolink PLA probe were added to the glass slide; the ligation mixture was then added to the surface of the glass slide. The signal was amplified and imaged with green-labeled oligonucleotide detection probes using a DeltaVision OMX V4 Blaze and a Leica SP8 fluorescence microscope. The implementation of negative controls of PLA was required. The main purpose was to verify the specificity of the antibody so that the images had no additional background. Briefly, only one antibody (Dynll1 or Cox4i1) was added in a glass slide, and the glass slide was incubated overnight at 4°C. Other procedures referred to the Duolink PLA.