Irdye 680cw donkey

Cells were harvested and lysed in RIPA buffer (R0278, Sigma.UK) with the addition of protease cocktail inhibitor (P8348, Sigma, UK), phosphatase inhibitor cocktail 2 (P5726, Sigma, UK) and phosphatase inhibitor cocktail 3 (P0044, Sigma) and stored at −20 °C. Proteins were quantified using BCA Protein Assay kit (23225, Thermofisher, UK). Samples were run on SDS-bolt gel (4–12%) bis-tris. Membranes were incubated with primary antibodies as follows: Mre11 (1:500, ab214), ß-actin (1:1000, ab8226), YY1 (1:1000, ab109228), GADPH (1:1000, ab9485), XRCC1 (1:1000, ab1838), pChk1 (1:1000, ab58567), Rad50 (1:1000, ab89), NBS1 (1:1000, Sigma, APREST78218), XRCC1 (1:500, ab1838), FEN-1 (1:1000, NB100-150), PARP (1:1000, 46D11 Cell Signaling), LIG1 (1:1000, ab177946), LIG3 (1:1000, HPA006723), RPA1 (1:1000, ab79398), RPA2 (1:1000, AB2175) and RPA3 (1:1000, ab97436). Membranes then were washed and incubated with Infrared dye-labelled secondary antibodies (LiCor) [IRDye 800CW Donkey Anti-Rabbit IgG (926-32213) and IRDye 680CW Donkey Anti-Mouse IgG (926-68072)] at dilution of 1:10,000 for 1 h. Membranes were scanned with a LiCor Odyssey machine (700 and 800 nm) to determine protein levels. Blots showed derived from the same experiments.

Proteins were analyzed by SDS-PAGE, followed by Western blotting and, for loading controls, Ponceaus S staining using standard methods.
For protein detection, mouse anti-opsin ([58 (link)] 1:1000), rabbit anti-emerin (Proteintech, 10351-1-AP; 1:1000), rabbit anti-LRRC59 (Sigma, HPA030829, 1:250), rabbit anti-calnexin (Enzo Life Sciences, Lörrach, Germany, ADI-SPA-860; 1:1000), rabbit anti-PDI (Cell Signaling, Cambridge, USA, 3501; 1:1000), mouse anti-TRC40 (Sigma, WH0000439M3-100UG; 1:1000), guinea pig anti-CAML (Synaptic Systems, Göttingen, Germany, 359004; 1:1000), rabbit anti-importin β ([61 (link)]; 1:1000), and rabbit anti-alpha-tubulin (Proteintech, 11224-1-AP; 1:1000) were used. For detection of secondary antibodies (LI-COR IRDye 800CW goat anti-mouse; IRDye 800CW donkey anti-mouse; IRDye 800CW donkey anti-rabbit; IRDye 680CW donkey anti-rabbit; IRDye 680CW donkey anti-mouse, IRDye 680CW donkey anti-guinea pig), the Odyssey system (LI-COR, Bad Homburg, Germany) was used. For comparison, the protein of interest was normalized to the signal of alpha-tubulin using Image Studio Lite 2.