Normal rabbit igg 2729

FA and methyl ferulate (MF) were purchased from Innochem Technology Co., Ltd. (Beijing, China). Compound C (CC) was purchased from Selleck Chemicals (Shanghai, China). CCl₄ and all cell culture supplemental components were purchased from Sigma-Aldrich (St. Louis, United States). HiScript III RT SuperMix cDNA reverse transcription Kits (R323-01) and AceQ^TM Universal SYBR qPCR Master Mix (Q511-02) were obtained from Vazyme Biotech (Nanjing, China). Antibodies against p-ERK1/2 (sc-7383), ERK1 (sc-271269), ERK2 (sc-81457), LKB1 (sc-32245) were purchased from Santa Cruz Biotechnology (Santa Cruz, United States). Antibodies against p-AMPK (ab23875), AMPK (ab131512) and p-LKB1 (ab63473) were from Abcam (Cambridge, United States). Antibodies against ALB (4929S), β-actin (4970S) and normal rabbit IgG (2729S) were obtained from Cell Signaling Technology (Danvers, United States). Antibodies against PTP1B (11334-1-AP) and FIBRONENCTIN (FN) (15613-1-AP) were purchased from Proteintech Group, Inc (Rosemont, United States). Goat anti-rabbit IgG-HRP (abs20040) and goat anti-mouse IgG-HRP (abs20039) were obtained from Absin Bioscience (Shanghai, China). Protein A/G-PLUS agarose beads (sc-2003) was purchased from Santa Cruz Biotechnology (Santa Cruz, United States).

HRECs and CSC complete recombinant medium were purchased from DS Pharma Biomedical Co. (Osaka, Japan). CAPE was purchased from Wako Pure Chemicals (Osaka, Japan). Anti­acetyl­histone H3 (#06-599), anti-acetyl-histone H4 (#06-598) rabbit polyclonal, and anti­actin mouse monoclonal (MAB1501) antibodies were purchased from Millipore Co. (Billerica, MA). Anti-HDAC1 (sc-7872), -HDAC3 (sc-11417) rabbit polyclonal antibodies and anti-MEF2 (sc-313), anti-MEF2X (sc-313X) rabbit polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). An anti-HDAC1 (#5356) mouse monoclonal antibody and normal rabbit IgG (#2729) were purchased from Cell Signaling Technology (Danvers, MA). An anti-MEF2D (610774) mouse monoclonal antibody was purchased from BD Transduction Laboratories (Lexington, KY). Horseradish peroxidase (HRP)-conjugated anti­rabbit (A6154) or ­mouse (A4416) IgG (whole molecule)-peroxidase antibodies were purchased from Sigma­Aldrich, Inc. (Saint Louis, MO). An anti-MEF2A phospho T312 (ab30644) rabbit polyclonal antibody was purchased from abcam (Cambridge, UK).

Coronal sections were rinsed with distilled water and stained with alum hematoxylin, followed by differentiation with 0.3% acid alcohol. After rinsing with distilled water, the sections were stained with eosin for 2 min and dehydrated for mounting. TUNEL assays (ThermoFisher Scientific) and Nissl staining (Servicebio, China) were performed according to the manufacturer’s instructions.
Immunostaining was performed using rabbit monoclonal anti-PARP16 (ARP33751, Aviva Systems Biology, USA) and anti-6E10 (800304, BioLegend). Normal rabbit IgG (2729, Cell Signaling Technology) was used as negative control. Secondary antibodies conjugated with horseradish peroxidase (ab64264, Abcam) were used and visualized according to standard protocols.

The following antibodies and reagents were purchased for use in the study: anti-α-smooth muscle actin antibody (α-SMA; A5228) from Sigma-Aldrich (St Louis, MO, USA); anti-MMP2 (#40994), anti-GAPDH (#5174), and anti-Tubulin (#2148) antibodies and normal rabbit IgG (#2729) from Cell Signaling Technology (Danvers, MA, USA); anti-COL1A1 (PAB17205), anti-Fibronectin (ab2413), anti-Timp1 (ab211926), and anti-TGFβ1 (ab179695) antibodies from Abnova (Colorado, USA) and Abcam; and TGFβ1 (240-B) from R&D Systems (Minneapolis, MN, USA). In addition, ABI Taqman primers/probes were purchased from Applied Biosystems (Foster City, CA, USA).

Immunoprecipitation was conducted to purify and enrich acetylated ERα to ensure the specificity and sensitivity of detection as described previously:^{15 (link),63 (link)} anti-acetyl Lysine (ab190479) antibody and protein A/G Sepharose (ab193262) from abcam (Cambridge, MA, USA); ERα (04-820) antibody from EMD Millipore (Billerica, MA, USA); normal rabbit IgG (#2729) from Cell Signaling Technology (Beverly, MA, USA).

Chromatin immunoprecipitation (ChIP) was performed following the instructions for SimpleChIP^® Enzymatic Chromatin IP Kit (Cell Signaling). For each immunoprecipitation performed, 1.6 × 10⁶ 3T3-L1 cells were seeded and grown to 2 days post–confluence (T0) before being treated, or not, with 1.0 uM DEX. After 20 h cells were fixed with 1% formaldehyde followed by glycine incubation. After cell lysis, chromatin was fragmented by micrococcal digestion and nuclear membranes were broken by sonication with a Bioruptor Sonicator (Diagenode). An aliquot of the digested chromatin (Input) was removed before sample immunoprecipitation with antibodies against either CEBPB (sc-7962, Santa Cruz Biotechnologies,) or a non-specific IgG control (Normal Rabbit IgG #2729, Cell Signaling). The complex co-precipitates were captured by Protein G magnetic beads and the cross-links were reverted before DNA purification. Chip-qPCR primers are listed in Table 1.

McCoy’s 5A modified medium (Mc5AM), Ham’s F-12 K (Kaighn’s) medium (F-12 K), Leibovitz’s L-15 medium (L-15), fetal bovine serum (FBS), Lipofectamine 3000, and Lipofectamine RNAi Max were purchased from Thermo Fisher (Carlsbad, CA). RPMI-1640 medium (1640) and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from GE Healthcare Inc. (Lafayette, CO). Antibodies against E2F1 (3742), stathmin1 (13655S), p-stathmin1 (Ser16) (4191S), TACC3 (8069S), p-TACC3 (Ser558) (8842S), clathrin (4796), Ki-67 (9449), and normal rabbit IgG (2729) were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against β-actin (E021020) and α-tubulin (E021030), and FLAG-tagged and HRP-conjugated secondary antibodies against rabbit IgG (E022230-01; E030120) and mouse IgG (E022060-01; E030110) were obtained from EarthOx (San Francisco, CA). 100 μM bucladesine (Selleck, Texas, USA, S7858) was used to activate PKA.