Normal serum

Uterine tissues were fixed overnight in 4% paraformaldehyde (PFD), prior to processing. Tissue processing include dehydration through increasing concentrations of ethanol, where the tissues were then cleared in chloroform and blocked in paraffin wax. Following these, tissues were cut into 5μm sections, deparaffinized in xylene and rehydrated in reducing concentrations of ethanol. Tri sodium citrate (pH 6.0) was used for antigen retrieval. Tissues were blocked in appropriate 10% normal serum (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature prior to incubation with CFTR, SLC26A6, SLC4A4-B, α-ENaC, β-ENaC, γ-ENaC and Na⁺/K⁺-ATPase primary antibodies (details shown in Table 1) at a dilution of 1:100 in PBS in appropriate serum. After three times rinsing with PBS, sections were incubated with donkey anti-goat IgG-FITC for CFTR and SLC26A6 (details in Table 1) at a dilution of 1:250 in PBS with 1.5% normal blocking serum at room temperature for 45 min. The slides were rinsed three times with PBS and were mounted with Ultracruz mounting medium (Santa Cruz Biotechnology, Santa Cruz, CA, USA), counterstained with DAPI to visualize the nuclei. All images were viewed under Nikon Eclipse 80i camera attached to the light microscope.

HEK293 cells were transiently co-transfected with FLAG-tagged α_1A – AR and one of the following GFP-tagged proteins: Rab5 (Rab5 Q79L) variant, Rab11 (Rab11 S25N) variant, β-arrestin-1 or β-arrestin-2. Rab5 and Rab11 were kindly provided by Dr. Marino Zerial (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany). Following serum-deprivation for 24h, cells were stimulated with 1 μM A-61603 or 1 mM ISO for 2h. After treatment, cells were fixed with freshly prepared 3.7% paraformaldehyde in PBS for 15 min at room temperature. Subsequently, cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 5 min, followed by nonspecific binding site blocking with 3% normal serum (Santa Cruz Biotechnology) in PBS for 1 h. Incubation with Alexa Fluor-568 conjugated anti-FLAG antibodies in blocking solution was done as directed by the manufacturer. Confocal microscopy was performed using a Zeiss LSM 510 META laser scanning microscope (Carl Zeiss, Inc., Thornwood, NY) equipped with a 60X objective, using the following laser wavelengths: excitation at 488 nm and emission at 505–530 nm; excitation at 543 nm and emission at 560–615 nm.

DCs (10⁷) were incubated with 10 μg/ml Rv2299c for 6 hr, and cell pellets were lysed with lysis buffer (10 mM Tris-HCl [pH 7.4], 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 μg/ml each aprotinin, leupeptin, and pepstatin, 1 mM Na₃VO₄, and 1 mM NaF). To prevent nonspecific binding, the cell lysates were precleared by adding 50 μl of normal serum (Santa Cruz) and 100 μl of 50% protein A or G Sepharose bead slurry (Invitrogen, Carlsbad, CA) to 1 mg of cell lysates. After 2-hr incubation at 4°C, the mixture of beads and cell lysates was centrifuged at 10,000 × g for 5 min at 4°C, and the supernatant was collected for the subsequent experiment. Rv2299c (His)-, TLR2-, and TLR4-associated proteins were immunoprecipitated by incubation with protein A or G Sepharose for 24 hr at 4°C after incubation with an anti-rat IgG Ab as a control Ab for anti-TLR2 and TLR4, an anti-mouse IgG Ab as a control Ab for the anti-Rv2299c (His) Ab for 1 hr at 4°C. The beads were harvested, washed and boiled in 5× sample buffer for 5 min. The proteins were separated by SDS-PAGE in a 10% gel followed by transfer of the proteins to a polyvinylidene difluoride membrane (Millipore). The membranes were further probed with anti-TLR2, anti-TLR4, and anti-His Abs as indicated.