Samples for analysis of the Stefin B expressing iMACs were prepared and the immunoblot performed as described previously 49 (link). Briefly cells were harvested, washed once in PBS and then lysed in RIPA lysis buffer (cOmplete protease inhibitors (Roche)), then sonicated to disrupt DNA to facilitate gel loading. The samples were then denatured and reduced by adding NuPAGE sample buffer (25% sample volume; Life technologies) and NuPAGE sample reducing agent (10% sample volume; Life technologies) and heating the samples to 95°C for 5 min. Proteins were separated by electrophoresis using a precast 4-12% SDS-PAGE gel (Novex, Invitrogen) and MES buffer (Novex, Invitrogen). The proteins were transferred to an Immobilon-FL PVDF membrane (Millipore) and blocked using 3% BSA in Tris-buffered saline pH7.4 (BSA-TBS) for 1h. The membrane was then probed with an anti-Flag antibody (Clone M2, Sigma) at a 1:1000 dilution and an anti-tubulin antibody (CST) at a 1:1000 dilution in BSA-TBS with 0.1% Tween overnight at 4C. Secondary antibodies (coupled to IRDye 680RD ot IRDye 800CW, Li-Cor Biosciences) were used 1:25,000 dilution.