Western blotting analysis of ICAM-1, p-JAK2, JAK2, STAT3, p-STAT3, SOCS1 and SOCS3

The cells were lysed on ice and centrifuged at 12,000 rpm and 4°C for 5 min. The supernatant was used for determination of the protein concentration with the bicinchoninic acid (BCA) method [5]. The protein samples were separated by 12% SDS-PAGE, electrotransferred onto PVDF membranes, blocked in Tris-buffered saline+Tween with 5% skim milk and then incubated overnight with antibodies against β-actin (Cell Signaling, Boston, MA, USA, 1:1000), ICAM-1 (1:1000), JAK2 (1:1000), p-JAK2 (1:200), STAT3 (1:300), p-STAT3 (1:1000), SOCS1 (Boster, Wuhan, China, 1:200) or SOCS3 (Boster, Wuhan, China, 1:200). After washing, the membranes were incubated for 2 h with HRP-labeled goat anti-mouse IgG or goat anti-rabbit IgG (Proteintech, Chicago, IL, USA) at room temperature. After washing, development, fixation and scanning, the image data were processed using ChemiDoc XRS (Bio-Rad Laboratories, Inc.). The relative level of p-JAK2 is presented as the gray-level p-JAK2/JAK2 ratio, and the relative level of p-STAT3 is presented as the gray-level p-STAT3/STAT3 ratio. The relative levels of the other target proteins are presented as the gray-level ratio of the target protein to β-actin.