Ha clone 16b12

Cells were lysed in 1× Laemmli buffer and the resulting whole-cell lysates were analyzed by Western blotting with the following primary antibodies: PML (Bethyl A301-167A), Flag (ABM G191 and Sigma F7425), GAPDH (Cell Signaling 14C10), vinculin (Sigma V9131), BLM (Bethyl A300-110A-M), RMI1 (Novus Biologicals NB100-1720), and HA (clone 16b12, Fisher Scientific NC9378714).

Immunofluorescence (IF) staining and IF-FISH were executed as described previously (Li et al. 2017 (link)). Primary antibodies used in this study were as follows: PML (Santa Cruz BIotechnology PG-M3), BLM (Bethyl A300-110A-M), Sp100 (Abcam ab43151), RPA (Thermo Scientific PA1-23299), Flag (ABM G191 and Sigma F7425), GFP (Invitrogen A6455), Myc (Cell Signaling 2276), TRF2 (Millipore 05-521 and Novus Biologicals NB110-57130), RMI1 (Novus Biologicals NB1001720), and HA (clone 16b12, Fisher Scientific NC9378714). Secondary antibodies used were Alexa 488, Alexa 555, and Alexa 647 (Molecular probes, Life Technologies). For IF-FISH, following the IF protocol, coverslips were denatured and hybridized TRITC-OO[TTAGGG]3-labeled PNA probe (PNA Bio). Digital images were taken using Zeiss Axio Imager M1. A minimum of 100 cells per condition were analyzed. A minimum of three independent experiments were performed unless otherwise indicated. Images were blinded and scored as following: Cells were defined as positive when at least five foci colocalized with telomeres.