Hla g

Cells were lysed using Laemmli Sample Buffer (Bio-Rad, 1610747) supplemented with 5% β-mercaptoethanol (MilliporeSigma, M3148). Lysates were heated to 95 °C for 10 min then loaded on 7.5%, 10%, or 12% SDS gel, depending on the size of target proteins, followed by transfer to PVDF membranes (MilliporeSigma, IPVH00010). Membranes were blocked in 5% skim milk (Bio-Rad, 1706404) or 5% BSA (Sigma-Aldrich, A7906) in TBS-T (Tris-buffered saline with 0.1% Tween 20), and incubated with primary antibody overnight at 4 °C, followed by washing three times in TBS-T. The membranes were incubated in secondary antibodies for 1 h at room temperature, followed by washing three times in TBS-T. Membranes were incubated with ECL Western blotting detection reagent (Fisher Scientific, 45-002-401) and imaged using ChemiDoc XRS+ (Bio-Rad). The following primary antibodies were used for Western blot: DLX5 (Novus Biologicals, NBP1-85793, 0.2 mg/mL), DLX6 (abcam, ab137079, 1:2,500), ASCL2 (R and D Systems, AF653, 1 ug/mL), ZNF439 (GeneTex, GTX119735, 1:1,000), NRIP1 (Millipore, MABS1917, 1:1,000), TFAP2C (Santa Cruz Biotechnology, sc-12762, 1:500), HLA-G (Santa Cruz Biotechnology, sc-21799, 1:500), MMP2 (Cell Signaling Technology, 40994, 1:1,000), and ACTB (Abgent, AM1829B, 1:20,000).

The paraffin sections were deparaffinized in xylene and rehydrated through graded alcohols to water. Next, antigen retrieval was performed by heating the sections in sodium citrate buffer at a high temperature for 10 min. Endogenous peroxidase activity was blocked by treating the sections with 3% H₂O₂. Then, nonspecific binding was blocked with 3% BSA (BIOFROXX, 4240GR100) for 1 h. The sections were then incubated overnight at 4°C with the appropriate primary antibody. The following day, corresponding secondary antibodies were added and incubated for 1 h at room temperature. After developing the color with DAB (ZSGB-BIO, ZLI-9017), the sections were counterstained with hematoxylin for 1–3 min. Finally, the sections were dehydrated and mounted with a xylene-based mounting medium, and images were captured using a microscope (Nikon).
The following antibodies were used: GPR65 (Abcam, ab188907, 1:100) and HLA-G (Santa Cruz, sc-21799, 1:100). MYLK (Proteintech, 21642–1-AP, 1:100), MYLK3 (Proteintech, 21527–1-AP, 1:100).