Ab76293

The methods for immunohistochemistry and in situ hybridization were described in our previous study^{20 (link),22 (link)}. For in situ hybridization, a probe for human miR-455-3p (Exiqon, Invitrogen, Shanghai, China) was used. For immunohistochemical analysis, deparaffinization and rehydration of the sections were performed with standard xylene-to-ethanol washes. The sections were then blocked in phosphate-buffered saline (PBS) plus 0.025% Tween 20 with 10% FBS. After blocking, the sections were incubated at 4 °C overnight with primary antibodies specific for PAK2 (1:100 dilution, Abcam, ab76293), phospho-PAK2 (1:100 dilution, Abcam, ab40795), COL2A1 (1:100 dilution, Abcam, ab188570), and MMP13 (1:80 dilution, Abcam, ab39012). Negative controls were prepared by substituting PBS for the primary antibodies. After overnight incubation, the sections were incubated with HRP-conjugated anti-rabbit IgG secondary antibody (Cell Signaling Technology, Boston, USA) for 30 min.

A549 cells transfected with lentivirus mediated sh-circ_0008717 or sh-NC were injected into the nude mice subcutaneously (4–6 weeks old). The tumor size was determined weekly. Mice were euthanized at day 28, and tumors were isolated, weighed, and used for the detection of circ_0008717, miR-1287-5p, and PAK2 using western blot or qRT-PCR. Also, tumors were fixed in formalin for immunohistochemistry (IHC) with the PAK2 antibody (1:250, ab76293, Abcam). All experiments were performed in line with the guidelines approved by the Ethics Committee of Tianjin First Center Hospital.

After 48 h of transfection, the cells were washed with pre-cooled PBS (Thermo Fisher Scientific, MA, USA) 3 times and then lysed in RIPA lysis buffer (Thermo Fisher Scientific, MA, USA) on ice for 10 min. The BCA assay kit from Thermo Fisher Scientific (MA, USA) was applied for quantification of the protein samples. After boiled at 95°C for 10 min, the protein samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 100V and then transferred to nitrocellulose membranes. Five percent BSA/TBST was used to block the membranes for 60 min. Subsequently, the membranes were incubated with primary rabbit polyclonal antibodies overnight at 4°C and with horseradish peroxidase (HRP) -conjugated secondary antibody goat anti-rabbit (ab6721, 1:3000, abcam, Cambridge, UK) in succession for 120 min of hybridization at room temperature. 1 × TBST (Solarbio, Beijing, China) was used to wash the membranes. Protein bands were visualized using the electrochemiluminescence kit (Solarbio, Beijing, China) and photographed. Primary antibodies include CXCL1 (PAI-29220, 1:2000, Thermo Fisher Scientific), Jak2 (ab108596, 1:5000, abcam), p-Jak2 (ab76293, 1:5000, abcam), STAT3 (9133S, 1:1000, Cell Signaling Technology), p-STAT3 (8204S, 1:1000, Cell Signaling Technology), and GAPDH (ab181602, 1:10000, abcam).