Normal rabbit igg

RIP-chip, that is, immunoprecipitation of RNA-induced silencing complexes (RISC) with an Ago2-specific monoclonal antibody followed by RNA extraction and subsequent quantification of mRNAs on microarrays, has recently been utilized to identify mRNAs that are associated with the RNA-silencing machinery and are therefore targets of cellular miRNAs [35 (link)–38 (link)]. In brief, 200 μg of total muscle protein was diluted with 200 μL of PBS buffer (pH 7.4). For each sample, 25 μL of protein A/G plus agarose (Santa Cruz) was washed with PBS and incubated with 2 μg of rabbit anti-Ago2 (Abcam, MA, USA) or rabbit normal IgG (Santa Cruz) antibodies for 2 h at 4°C. The beads containing the immobilized anti-Ago2 antibody were then added to 400 μL of diluted serum and incubated for 4 h at 4°C. The beads were washed 3 times with 1% NT-2 buffer (1% Nonidet P-40, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 2 mM EDTA) and the mixture was split in half. One-half of each sample was eluted in 2 × SDS sample buffer and subjected to SDS/PAGE and immunoblotting with a mouse anti-Ago2 antibody (Santa Cruz, CA, USA) to detect Ago2. The other half of each sample was eluted in 600 μL of lysis/binding buffer from the mirVana miRNA Isolation Kit (Life Technologies, NY, USA) and processed for RNA isolation. The RNA pellet was used for oligo-dT purification and library generation.

Whole proteins of liver tissue were extracted and measured protein concentrations using the bicinchoninic acid (BCA) protein assay. Part of the supernatant was used as input control and the rest was immunoprecipitated overnight at 4ºC by gently rocking with anti-HK2 antibody (Abcam Inc., Cambridge, UK).
Approximately 5 μl antibodies were used for 500 μg total protein. Then protein A/G agarose beads (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) were added to bind with the immunoprecipitates for 2 hr with gentle shaking at room temperature. Precipitated proteins were washed 3 times with PBS and boiled with 5×loading buffer, then immunoblotting was performed as previously described. Rabbit normal IgG (Santa Cruz Biotechnology Inc.) served as negative control.

ChIP analysis was performed as previously described [52 (link)]. Briefly, EpH4 cells were cross-linked with 1% formaldehyde for 10 min at room temperature. For each group, a 10-cm dish of EpH4 cells with 80% confluency were lysed and chromatin was sonicated in a sonicator for 30 min (with 7-sec sonication and 7-sec rest alternatively). Sonicated chromatin was then diluted and immunoprecipitated with anti-E2F1 IgG or rabbit normal IgG (Santa Cruz, sc-2027). Immunoprecipitation products and input were analyzed by quantitative PCR using the following specific primers:
Site a,
forward:5’-CAGCTCGAGATGAATGGAAACC-3’,
reverse: 5’-TAGCCAATCAGACCCGGACTTC-3’.
Site b,
forward: 5’-CAGCAATGTCTCCATGTCACAT-3’,
reverse: 5’-CCCATGAGCACAACACAATTTC-3’.

The whole proteins of cardiomyocytes were extracted using the special lysis buffer for immunoprecipitation. The lysates were centrifuged, and protein concentrations were measured by BCA assay. Part of the supernatant was used as input control, and the rest was immuneprecipated overnight at 4°C by gently rocking with anti‐ULK1 antibody. Approximately 4 μL antibodies were used for 400 μg total protein. Then, protein A/G agarose beads (Santa Cruz) was added to bind with the immunoprecipitates for 2 hours with gently shake at room temperature. Precipitated proteins were washed 3 times with lysis buffer and boiled with 5 × loading buffer, and immunoblotting was performed as previously described. Rabbit normal IgG (Santa Cruz) served as negative control.

Pierce Co‐Immunoprecipitation Kit (Thermo Scientific, Waltham, MA, #26149) was used to carry out the immunoprecipitation studies. Briefly, RPE‐choroid preparations from seven mice of each genotype were sonicated in IP Lysis/Wash Buffer (provided in the kit) plus 1% protease inhibitors (Sigma). The total lysates were processed with the kit according to the instructions. Seventy micrograms of lysates of each genotype were immunoprecipitated with 10ug immobilized βA3/A1‐crystallin antibody at 4°C overnight. Normal rabbit IgG (Santa Cruz, sc‐2027) was the negative control. Samples from elution were loaded for SDS‐PAGE analysis. Fifteen micrograms of RPE‐choroid lysates of each genotype were loaded as the input for the SDS‐PAGE analysis.

ChIP assays were performed as previously described [73 (link)]. The antibodies used for immunoprecipitation were: normal rabbit IgG (Santa Cruz), polyclonal antibody for PARP-1 (Enzo Lifescience), H3Ac (Millipore), H3K4me3 (Millipore) and H3K27me3 (Millipore). The sense oligonucleotide used to amplify the promoter region was 5′-AACCCCGCCTCGGAGGAGT-3′ and the antisense oligonucleotide was 5′-CCAATCGGAGGCTCGTCT-3′. Real-Time PCR assays were carried out to amplify the enhancer region as already described [62 (link)]. The sense oligonucleotide used was FW 5′-GAGCAGCCCTTAATGACTTG-3′ and the antisense oligonucleotide was 5′-CCCAACTCCCTAACTTCCC-3′. The sense oligonucleotide used to amplify the promoter region of ITPR1 was 5′-ACTGAGGTCGCGGTTTGTAT-3′ and the antisense oligonucleotide was 5′-AAGGAGCCGTGTTGTGACTT-3′ [63 (link)].