Nf κb p100 52

Cells were lysed in SDS lysis buffer (100 mM NaCl, 500 mM Tris, pH 8.0, 10% SDS). For detection of NF-κB p100/52, p65 and c-Rel, a nuclear extraction kit (Thermo Scientific, MA, USA) was used to separate nuclear and cytoplasmic fractions. Cell extracts were subjected to 8–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies for Western blotting include: Galectin-3 (cat. 125402) (Biolegend, San Diego, USA), Galectin-1 (cat. Ab25138) and TSG101 (cat. ab30871; detects murine and human proteins) (Abcam, Cambridge, MA, USA), CD63, Erk1/2, NF-κB p65 (sc-8008 Figure 4G) (Santa Cruz Biotechnology, USA), phospho-Erk1/2, NF-κB p65 (cat. 8242) and calreticulin (cat. 2891S; detects murine and human proteins) (Cell Signaling Technology, USA), NF-κB p100/52 (cat. 06–413) (Millipore, USA). Gapdh (Chemicon International, USA or Millipore MAB374), α-tubulin (Oncogene Science, Cambridge, USA), lamin A, Histone H3 (Cell Signaling Technology, USA) or β-actin (cat. GTX109639, GeneTex) were used as a loading control.

RNA was isolated from the cells or mouse prostate tissues using Rneasy Kit (Qiagen) with the addition of Dnase I to minimize DNA contamination. Imprinting was performed using a FluPE assay as previously described [6] (link). For human IGF2, a single nucleotide polymorphism identified on IGF2 exon 7 (G/C) was used to identify individual alleles. IGF2 imprinting was examined on Exon 6 (A/G) in mouse prostate tissues. Differences were determined by calculating the ratio of their respective spectral intensities [repressed allele (Ai)/active allele (Aa)]. Quantitative PCR was performed using an iCycler (Bio-Rad) and SYBR Green PCR master mix (Applied Biosystems) to measure gene expression, primers are available on request. Western blot was performed to detect protein expression using antibodies for CTCF (Cat #07-729, Millipore), NF-κB p50 (4D1, Santa Cruz), NF-κB p65 (c-20, Santa Cruz), NF-κB p100/52 (18D10, cell signaling), IKKα/β (sc-7607, Santa Cruz) and IκBα (c-21, Santa Cruz) and α-Tubulin (DM1A, EMD).

30μg total tissue protein extracted from mouse kidney was separated on 8–12% SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane pre-activated by methanol in the transferring buffer. Membranes were blocked with 5% skimmed milk for 30min, and were incubated 2-hour with specific primary antibodies at room temperature. Immunoreactive bands were detected using HRP-conjugated goat anti-rabbit or goat anti-mouse IgG as the secondary antibody (1:5000) (Santa Cruz). Primary antibodies included antibodies against Caspase1 p10 (GeneTex), p-JNK, NF-κB p100/52, p-IκBα and tubulin (Cell Signaling).