Newblot nitro stripping buffer

HUVECs were transfected with miRNAs or siRNAs as described above. Forty-eight hours after transfection, the cells were treated with 10 ng/ml of IL6 (Cell Signaling Technology) or 1000 U/ml of IFNαA (Sigma) for 20 minutes after which cells were washed once with ice-cold PBS and harvested in RIPA buffer. Erythropoietin treatments of HUVECs were performed with 250 U of EPO (Sigma) for 10 minutes. Equal amount of lysates were used for Western blot analysis to measure levels of pY705-STAT3, pS727-STAT3, STAT3, PKCδ, IRAK1, pY701-STAT1, pY694-STAT5 or STAT5 and their levels normalized to that of actin. When necessary, blots were stripped of their primary antibody using the Newblot Nitro Stripping buffer (Li-COR) and reprobed.

Protein concentration was determined by a modified Lowry method (Markwell et al. 1978 (link)). This modified method utilizes the detergent deoxycholate to solubilize hydrophobic proteins present in membranes. The mitochondrial fractions were added to loading buffer (0.05% bromophenol blue, 25% glycerol, 6% SDS, 6 mM EDTA, and 150 mM Tris-HCl pH 8.8 and 0.5% β-mercaptoethanol), and incubated for 1 hr at 37°. Samples were resolved by 12% SDS-PAGE and the separated proteins were transferred to a nitrocellulose membrane. The membranes were incubated for 1 hr under constant shaking in Odyssey blocking buffer (LI-COR). The blots were then incubated with an appropriate dilution of primary antibody (Table S2 in File S1) in blocking buffer under constant shaking overnight at 4°. The following day, the membranes were washed three times for 10 min each with 0.1% Tween-20 in phosphate-buffered saline (PBS), and were then incubated with an appropriate dilution of secondary antibody in LI-COR blocking buffer for 1 hr. Excess antibody was washed off with 0.1% Tween-20 in PBS and proteins were detected and imaged by using a LI-COR Odyssey Infrared Imaging System. The membranes were stripped with New Blot Nitro Stripping Buffer (LI-COR) for 10 min under constant shaking at room temperature, and reprobed with anti-porin antisera (Por1) as a loading control.

HBMEC cultures were washed with PBS, and protein was harvested by lysing the cells with Radio Immuno Precipitation Assay (RIPA) buffer containing Complete Mini Protease Inhibitor Cocktail (Roche). HMC3 were washed with PBS, with additional steps performed to isolate the nuclear and cytoplasmic fractions as described earlier [28 (link)].
Protein concentrations were quantified using a BCA Protein Assay Kit (ThermoFisher Scientific, ref #23223) and immunoblotting was performed as described previously [6 (link)]. The following primary antibodies were used: anti-gp130 (IL6ST; Abcam, ab283685), anti-IL-6R (Abcam, ab128008), anti-phosphorylated STAT3 (Tyr705, Cell Signaling, 9145T), and anti-STAT3 (Cell Signaling, 124H6). In addition, the following secondary antibodies (all from Licor) were applied: IRDYE^® 680RD (926-68073 + 926-68072) and IRDYE^® 800CW (926-32212 + 926-32211).
Protein levels were normalized to anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Novus, NB3000-221R) for total and cytoplasmic fractions, and to anti-Lamin A/C (Cell Signaling, #4777S) for nuclear fractions. Membranes that were used to read multiple proteins were stripped using NewBlot Nitro Stripping Buffer (Licor, ref #928-40030) for 10 min, then the steps were repeated starting with the blocking of the membrane.

Vaginal tissues were removed at 0 and 1 dpi, snap frozen in liquid nitrogen, and then homogenized. Homogenized tissue was then lysed in 1mL of cold radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors. Protein concentration was then determined using a Bradford Assay. The immunoblot procedure was then conducted as previously described [72 (link)]. Briefly, 15–20 μg of protein was loaded on to a 10% SDS-Page Gel and run at 95V for 12 min, and 1 hr for 120V. Gels were transferred onto a nitrocellulose membrane for 1 hour at 400 mA. The membranes were then blocked with LI-COR Odyssey blocking buffer (Mandel). Blots were probed for anti-mouse IL-33 (R&D, AF3626) using a 1:1000 dilution and anti-actin (Santa Cruz Biotechnology, sc-1616) at 1:2000 at 4°C overnight. Blots were stripped with LI-COR NewBlot Nitro Stripping Buffer. Primary antibodies were detected using an anti-goat IRDye infrared secondary antibody at a 1:10 000 dilution and then imaged with the LI-COR Odyssey infrared scanner. Band densitometry was assessed using software from Image Studio Lite (LI-COR). IL-33 bands were normalised to actin levels, and then quantified as fold changes to d0 vaginal tissue samples.

BmK venom was purchased from a domesticated scorpion farm (Kaifeng, China), where it was collected by electrical stimulation. Sephadex G-50 and CM-Sephadex C-50 were purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). Acetonitrile (HPLC grade) was from Tedia (Cincinnati, OH, USA). Dialysis membrane (500 Da cut off) was a product of Minnesota Mining and Manufacturing (St. Paul, MN, USA). Trypsin, L-glutamine, fetal bovine serum, Neurobasal medium, Hoechst 33,342, and anti-MAP2 primary antibody were obtained from Life Technology (Grand Island, NY, USA). Primary antibodies against Akt, phosphorylated (p)-Akt, were obtained from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies and NewBlot Nitro Stripping Buffer were from LI-COR Biotechnology (Lincoln, NE, USA). Trifluoroacetic acid, cytosine arabinoside, poly-L-lysine, N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), GW 441756, and all inorganic salts were obtained from Sigma-Aldrich (St. Louis, MO, USA). Tetrodotoxin was purchased from Tocris Bioscience (Ellisville, MO, USA). The Ca²⁺-specific fluorescent dye Fluo-4/AM was obtained from AAT Bioquest (Sunnyvale, CA, USA). NGF ELISA kit was purchased from Kete Biological Technology Co., Ltd. (Nanjing, China).