Mms 407r

Whole cell protein extraction was accomplished by TCA precipitation as previously described and fractionated by SDS-PAGE [89 (link)]. Western blots were probed using the following antibodies; anti-PCNA at 1:4000 dilution (S871, a gift from B. Stillman, CSHL), anti-SUMO at 1:3000 dilution (A gift from X. Zhao, MSKCC), anti-ubiquitin at 1:1000 dilution(P4D1, Covance), anti-Rad53 at 1:1000 dilution (A gift from JFX Diffley, LRI, UK), anti-phospho-S129 H2A at 1:1000 dilution (ab15083, Abcam), and anti-tubulin at 1:5000 dilution (MMS-407R, Covance).

Cell lysates from 293T cells, transiently transfected with each HA-tagged A3 protein or the expression vector alone, were resolved by 12% polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difloride membrane (PVDF, MilliporeSigma). Membranes were probed with the test bleeds (1:1000), cell-free supernatants from each hybridoma cell line (1:3), purified 5210-87-13 mAb (1:2000), anti-HA (C29F4, #3724, Cell Signaling Technology, Danvers, MA, USA, 1:1000), or anti-tubulin (MMS-407R, Covance, Emeryville, CA, USA, 1:20,000) in 50% BLOK (MilliporeSigma) and 0.1% Tween 20 in PBS. The membranes were then incubated with secondary antibody, either goat anti-rabbit-HRP (1:5000 Jackson ImmunoResearch Laboratories Inc.), anti-rabbit IgG IR800CW (Odyssey 926-32211, 1:20,000, LI-COR, Lincoln, NE, USA), or anti-mouse IgG IR800CW (Odyssey 827-08364, 1:20,000) in 50% BLOK (MilliporeSigma) and 0.1% Tween 20 in PBS. A subset of immunoblots used Abcam 1184990 (1:2500, Cambridge, MA, USA) and Proteintech 14559-1-ap (1:500, Proteintech Group Inc., Rosemont, IL, USA) mAbs diluted in PBS, supplemented with 5% milk protein and 0.1% Tween 20. Signals were detected with HyGlo (Thomas Scientific, Swedesboro, NJ, USA) on film or imaged using LiCor.

Transmission electron microscopy was performed as previously described (36 (link)). Hematoxylin & eosin staining was carried out as described (37 (link)). For immunostaining, embryos or tissues were fixed with 4% paraformaldehyde overnight at 4°C and then rinsed with PBS 3 times. The samples were dehydrated in an increasing gradient of 10% to 30% sucrose/PBS and embedded in OCT (Tissue Tek). The samples were cryosectioned at 10-μm thickness, and OCT was washed out by PBS. The sections were incubated with diluted primary antibody overnight at 4°C and then with cy2- or cy3-labeled secondary antibody (Jackson Immunoresearch) for 1 hour at room temperature with PBS wash 3 times in between. The following antibodies were used for immunostaining: rabbit-anti-Numb (Cell Signaling, 2756s, 1:200 dilution), mouse anti-α-Actinin (Sigma, A7811, 1:500 dilution), mouse anti-α-Actin (Sigma, A9357, 1:500 dilution), goat anti-NEBL (Abcam,ab99420, 1:100 dilution), mouse anti-α-Tubulin (Covance, MMS-407R, 1:800 dilution), mouse anti-Desmin (Covance, MMS-454s, 1:200 dilution), and Phalloidin (Invitrogen, A12379 1:500 dilution).