Hrp conjugated goat anti rabbit igg

Primary antibodies: mouse monoclonal anti-human HIF-1α (1:250, ref. 610959 BD Transduction Laboratories); goat polyclonal anti-human actin (1:1000, ref. sc1615 Santa Cruz Biotechnology); in house mouse monoclonal anti-human carbonic anhydrase IX- M75 hybridoma medium (1:3); rabbit polyclonal anti-human ADCY VI (1:250 in WB, ref. SAB2100054 Sigma); rabbit polyclonal anti-human ADCY VI (1:50 in IF and FACS, ref. sc25500 Santa Cruz Biotechnology); goat polyclonal anti-human ADCY VII (1:500 in WB, ref. sc32120 Santa Cruz Biotechnology), rabbit polyclonal anti-human ADCY VII (1:50 in IF and FACS, ref. SAB1303163 Sigma); rabbit monoclonal anti-human phospho-PKA substrates (1:1000, ref. 100G7E Cell Signaling); mouse monoclonal anti- cAMP (ref. ab24851 Abcam). Secondary antibodies: Alexa Fluor 488-conjugated donkey anti-mouse IgG; Alexa Fluor 488-conjugated donkey anti-rabbit IgG; (both 1:1000, Invitrogen); HRP-conjugated goat anti-mouse IgG (1:5000 in Western blotting, 1:2000 in ELISA, Dako); HRP-conjugated rabbit anti-goat IgG (1:5000, Dako); HRP-conjugated goat anti-rabbit IgG (1:5000, Dako).

Twenty micrograms of total protein lysates was size-fractionated on 4 to 12% NuPAGE Bis-Tris Gel (NP0321, Invitrogen). OLR1 was detected by incubation with a rabbit anti-mouse OLR1 antibody (1:500, ab203246; Abcam), followed by HRP-conjugated goat anti-rabbit immunoglobulin G (IgG) (1:1000; P0448, Dako). LDLR was detected by incubation with a rabbit polyclonal antibody (1:1000; 3839-100, BioVision, USA) at 4°C overnight followed by HRP-conjugated goat anti-rabbit IgG (1:1000; P0448, Dako). α-Tubulin was used as a housekeeping control (sc-32293, Santa Cruz Biotechnology). Detection of immunoreactive products was performed using 1:1 ECL reagent (RPN2235, GE Healthcare) and a C-DiGit Blot scanner (Model 3600, LI-COR). Densitometry was performed with Image Studio Lite software (LI-COR).

Total RNA was isolated immediately after the cells were harvested using Trizol (Invitrogen, 15596-018) according to the manufacturer's instructions; mRNA was prepared from total RNA using the Dynabeads mRNA purification kit (Ambion, catalog no. 61006). The concentration of purified mRNA was determined with a NanoDrop. RNA samples were immobilized twice using UV cross-linking (Stratalinker 2400) and were incubated at 80°C for 1 h. The blot was blocked using blocking buffer (PBS, 10% SDS, 1 mM EDTA) for 20 min. The mRNA was used for dot blot analysis using an antibody specific for m⁶A (1:1,000; Synaptic Systems; catalog no. 202003). The membrane was incubated with HRP-conjugated Goat anti-rabbit IgG (DakoCytomation, p0448, 1:10,000 dilution; 20 ng/ml) in 10 ml of antibody dilution buffer for 1 h at room temperature. The membrane was washed four times for 10 min each in 10 ml of wash buffer. To visualize the spots, the membrane was incubated with 3 ml of ECF substrate (GE Healthcare) for 5 min in darkness at room temperature. The intensity of the dot blot signal was analyzed by Image J software.