Western Blot Analysis of WRN Protein

Log phase LCLs (~10⁶ cells/ml) were harvested, washed 1× in PBS, and flash frozen in liquid N₂. Cell pellets were lysed in high salt buffer as described previously28 (link), and protein concentrations were measured by the Bradford assay. Approximately 100 μg of total protein were electrophoresed through a NUPAGE 4–12% Bis-Tris polyacrylamide gradient gel (Life Technologies) following the manufacturer’s guidelines. The gel was then transferred overnight to a PVDF membrane (Immobilon-FL, Millipore) using Tris-glycine-methanol transfer buffer. Non-specific membrane interactions were minimized by incubation in a 5% milk solution prepared in TBS-T (Tris-buffered saline, pH 7.5 + 0.05% Tween 20). The membrane was incubated overnight at 4 °C with a WRN- specific mouse monoclonal antibody (clone 195C; Sigma; 1:1000)40 (link) and simultaneously with mouse anti-nucleolin antibody (Invitrogen; 1:1000) to control for loading differences. The membrane was incubated with AlexaFluor 647 anti-mouse antibody (Invitrogen; 1:1000) for 1 hr at room temperature, washed three times in TBS-T, and then imaged on an AlphaInnotech FluorChemQ Imaging Station. Band intensities were quantified using ImageJ software.