Ab228748

RIPA lysate was used to prepare tissue homogenate to extract protein and NP-40 lysate to obtain cell protein. The protein concentration was measured using the BCA protein kit (TaKaRa, Otsu, Japan, T9300A). Protein was degenerated in sodium dodecyl sulfate (SDS) buffer, followed by separating on SDS-polyacrylamide electrophoresis (PAGE) gel using electrophoresis. After that, they were transferred onto polyvinylidene difluoride (PVDF, Millipore, USA, IPVH00010) membrane and incubated with primary antibody actin (Abcam, USA, ab8226, 1 : 400), BAX (Abcam, USA, ab32503, 1 : 200), Bcl-2 (Abcam, USA, ab182858, 1 : 200), caspase-3 (Abcam, USA, ab32351, 1 : 1000), Cbfα-1 (Abcam, USA, ab113203,1 : 200), OPN (Abcam, USA, ab228748, 1 : 200), SM-α (Abcam, USA, ab7817, 1 : 500), Akt (Abcam, USA, ab8805, 1 : 200), p-Akt (Abcam, USA, ab38449, 1 : 100), ERK1/2 (Abcam, USA, ab184699,1 : 1000), p-ERK1/2 (Abcam, USA, ab278538,1 : 1000), and β-actin (Abcam, USA, ab8226, 1 : 1000) overnight at 4°C. Next, they were incubated with horseradish peroxidase- (HRP-) conjugated secondary antibodies IgG (Abcam, USA, ab150077, goat anti-rabbit, 1 : 5000) at room temperature for 45 min. After the membrane was washed in TBST, the blot was visualized by the enhanced chemiluminescence ECL kit (Thermo Fisher Scientific, NY, USA).

The skin explants were fixed with neutral-buffered 10% formalin overnight, washed with PBS, and then embedded in paraffin. Sections were cut into 3-µm thick slices, deparaffinized in xylene, hydrated in a graded series of ethanol, and subjected to antigen retrieval by the microwave method with EDTA. The samples were blocked with 10% bovine serum albumin and 0.5% Tween-20 in PBS. The slides were incubated overnight with primary antibodies at 4 °C. Incubation with fluorescence-labeled secondary antibodies was performed for 30 min at 37 °C. The following primary and secondary labeled antibodies were used: rabbit polyclonal to OPN3 N-terminal (ab228748; Abcam) conjugated to Alexa Fluor 488-labeled goat anti-rabbit IgG (A0423; Beyotime); mouse anti-human pan-cytokeratin monoclonal antibody (sc-81703; Santa Cruz Biotechnology, Inc.) conjugated to Cy3-labeled goat anti-mouse IgG (A0521; Beyotime); rabbit anti-human Dync1i1 polyclonal antibody (AB_2846296, Affinity Biosciences, Ltd.) conjugated to an Alexa Fluor 488-labeled goat anti-rabbit IgG (A0423; Beyotime); and rabbit anti-human DCTN1 polyclonal antibody (AB_2838577, Affinity Biosciences, Ltd.) conjugated to Alexa Fluor 488-labeled goat anti-rabbit IgG (A0423; Beyotime). Nuclear staining was performed with DAPI. Fluorescent images were collected by fluorescence microscopy (Zeiss, Oberkochen, Germany).