P akt d9e

Immunofluorescence analysis was performed as described previously [21 (link)]. Briefly, whole ovaries and fallopian tubes were fixed in 10% formalin and embedded in paraffin. Antibodies for K8 (904801, 1:1000, BioLegend, San Diego, CA, USA), YFP/GFP (Ab290, 1:200, Abcam, Cambridge, MA, USA), Tomato (Ab62341, 1:300, Abcam), p-AKT (D9E, 1:100, Cell signaling, Danvers, MA, USA), PAX8 (10336-1-AP, 1:1200, Proteintech, Rosemont, IL, USA), ERα (SC542, 1:50, Santa Cruz, Dallas, TX, USA), and β-catenin (610154, 1:200, BD bioscience, Billerica, MA, USA) were used. Antigen retrieval (Citrate buffer pH 6.0, 20 min boil in a microwave oven) was performed prior to incubation with the above-listed antibodies.

HIV-1_BaL was generated in CD8-depleted PBMCs in the presence of 10 nM retinoic acid (Sigma-Aldrich, St Louis, MO), 20 U/ml IL2 (NIH) and 50 ng/ml of anti-CD3 monoclonal antibodies (clone OKT3; e-Bioscience, San Diego, CA) and titered in TZMbl cells as previously described^{14 (link)}. Lamivudine (3TC) (Sigma-Aldrich) was used as a control for inhibiting HIV-1_BaL infection. PBMCs and endocervical tissues were incubated with E2 (Sigma Aldrich). The following inhibitors or activators of E2 signaling were used: selective estrogen receptor modulator (SERM) Raloxifene (Sigma-Aldrich), and LIMKi3 (LIMK1/2 inhibitor blocking CFL1 phosphorylation; Tocris, Minneapolis, MN).
Antibodies (Abs) used in Western Blot were rabbit monoclonal anti-human CFL1 (D3F9), pCFL1 (77G2), β-Actin (D6A8); AKT (pan) (C67E7) and pAKT (D9E) (Cell Signaling Technology, Danvers, MA); HPRT1 (Sigma-Aldrich); and HRP-conjugated goat anti-rabbit IgG (secondary Ab) (Abcam, Cambridge, MA).
Antibodies used for Immunofluorescence staining (IF) were rabbit monoclonal anti-human CFL1 (EP6376) (Abcam); mouse monoclonal HIV-1-p24 (24-4)-AlexaFluor 488 (AF488) (Santa Cruz Biotechnology); mouse monoclonal anti-human CD3 (OKT3)-AF488, IgG-AF488 (eBM2a), goat anti-rabbit secondary Ab AF568, IgG isotype controls (Invitrogen, Carlsbad, CA) and DAPI (Sigma-Aldrich). FVS 780-APC-Cy7 (FVS780) (Live/Dead dye) was used for flow cytometry.

Harvested cells were washed with PBS and lysed in PhosphoSafe (Novagen) following the instructions of the manufacturer. Total protein concentrations were quantified (Bradford reagent) and protein isolates were loaded on 12.5% SDS-PAGE gels and later transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked using block-buffer (Invitrogen) and incubated at 4°C with the primary antibody. We used the following antibodies: PIK3R1 (N-term L11, anti-Rabbit, Sigma Aldrich), Akt 1/2/3 (5C10, anti-Mouse, Santa Cruz), p-Akt (D9E, anti-Rabbit, Cell Signaling), STAT3 (EPR787Y, anti-Rabbit, Abcam), p-STAT3 (EP2147Y, anti-Rabbit, Abcam), cleaved PARP1 (E51, anti-Rabbit, Abcam). ERK-2 (C14, anti-Rabbit, Santa Cruz) was used as loading control. We utilised horseradish peroxidase–coupled secondary antibodies and the ECL Plus system (GE Healthcare) to visualize the protein expression. All experiments were repeated at least three times.