Gfp jl 8

These were carried out as described previously [18 (link), 21 (link)]. Western blotting experiments were usually performed 2–3 times, and representative results are presented in figures. The following primary antibodies were used: ATF3 (sc-188X, 1:10,000), MDM2 (N-20, sc-813, 1:1000), p53 (DO-1, sc-126, 1:1000), and HA (sc-7392, 1:2500) from Santa Cruz Biotechnology; PUMA (2–16, 1:1000) from CALBIOCHEM; p21(556,431, 1:1000) from BD Pharmingen; β-actin (A2228, 1:10,000) and FLAG (F3165, 1:5000) from Sigma; and GFP (JL-8, 1:8000) from Clontech.

HEK 293 T cells were transiently transfected with 600 ng DNA in a 12-well format using the PEI transfection reagent. After 48 h, cells were lysed in Laemmli sample buffer. Total cell lysates were analyzed through immunoblotting using anti-menin (A300-105A, Bethyl) and anti-alpha tubulin (CP06 Calbiochem) antibodies respectively. In addition, total RNA was isolated and quantitative RT-PCR was performed to determine total MEN1 and ACTB (beta-actin) expression in pCDNA3.1 MEN1 transfected cells as described [47 (link)]. Menin protein levels were determined using ImageJ [48 ] from three independent transfections after subtraction of the background/menin signal from empty vector transfected cells. Ratios of relative menin protein and MEN1 mRNA levels determined in triplicate were calculated.
For doxycycline-inducible expression in stable cell lines MEN1-GFP expressing cDNAs were chromosomally integrated in HeLa cells using the Flip-in system, essentially as described [10 (link)]. After 24 h of induction using 1 µg/ml doxycycline, GFP expression was verified using immunoblotting using GFP (JL-8- Clontech) and α-tubulin (CP06 Calbiochem) antibodies. Nuclear and cytoplasmic extracts were prepared for GFP-affinity purification coupled to mass spectrometry analyses as described before [10 (link)].

Total cell lysates were collected for Western blot analysis or coimmunoprecipitation assay as described [17 (link)]. Cell fractions were collected using Nuclear Extraction Kit (SK-0001, Signosis, Santa Clara, CA, USA) according to the manufacturer’s instructions. The antibodies used in this study are described below: GFP (JL-8, Clontech, Mountain View, CA, USA), STAT3 (9139, Cell signaling, Danvers, MA, USA), phospho-STAT3/Y705 (9145, Cell signaling), GAPDH (sc32233, Santa Cruz, Dallas, TX), and α-tubulin (DM1A, T6299, Sigma-Aldrich, St. Louis, MO, USA). The CPAP polyclonal antibody was the same as that used in our previous studies [17 (link)]. Protein signals were detected using secondary HRP-conjugated anti-mouse or anti-rabbit antibodies by the Western Lightning Plus-ECL system (PerkinElmer, Waltham, MA, USA).

For the detection of FAM188B, we generate polyclonal antiserum using FAM188B protein C-terminus peptides (722–738 a.a: TISEDTDNDLVPPLELC) as antigen (AbFrontier, Seoul, Korea) (Supplementary Figure 2). The affinity-purified serum was applied to immunoblots at 1:1000 dilutions in 5% skim milk/1× Tris-buffered saline (TBS). The following antibodies were used to detect other proteins in this study: FLAG M2 and β-actin from Sigma-Aldrich (St. Louis, MO, USA); PUMA, USP7, p21, BAX and PARP from Cell Signaling Technologies (Danvers, MA, USA), and p53 (DO-1), p53 (FL393), phospho-p53 (ser15), and α-tubulin from Santa Cruz Biotechnology (Dallas, TX, USA), and GFP (JL-8) from Clontech (Mountain view, CA, USA).