Invitrogen ibright imaging systems 1500

The total protein extraction kit for cultured animal cells and tissues (Invent Biotechnologies, Inc.) was used for protein extraction from HNSCC cell lines. Protein extracts were electrophoresed using a 4%–12% NuPAGE Bis‐Tris SDS‐PAGE gel (Invitrogen, Thermo Fisher Scientific Inc.) and transferred onto an Immobilon‐P membrane (Merck Millipore).
The membrane was then incubated with mouse anti‐human HOXB7 Ab (40–2000, 1:100 dilution, Invitrogen), rabbit anti‐phospho‐p44/42 MAPK (pERK1/2) Ab (Thr202/Tyr204; Cell Signaling Technology), rabbit anti‐p44/42 MAPK (ERK1/2) Ab (137F5; Cell Signaling Technology), mouse anti‐CIITA Ab (7‐1H; Santa Cruz Biotechnology), rabbit anti‐phospho‐Stat1 Ab (D4A7; Cell Signaling Technology), rabbit anti‐Stat1 Ab (42H3; Cell Signaling Technology), and mouse anti‐human β‐actin Ab (C4; Santa Cruz Biotechnology). The Amersham ECL Prime Western Blotting Detection System (GE Healthcare Life Sciences) and Invitrogen iBright Imaging Systems 1500 (Invitrogen) were used for detection by chemiluminescence. ImageJ was used to analyze the expression values of the target bands.²⁵

The tumor cell line proteins extracted using the Miute^TM Total Protein Extraction Kit (Invent Biotechnologies, Inc.) were subjected to electrophoresis on NuPAGE Bis–Tris gels (Invitrogen, Thermo Fisher Scientific Inc.) and transferred to an Immobilon-P membrane (Merck Millipore). The membrane was incubated with mouse anti-human FGFR1 Abs (M19B2, Novus Biologicals) and mouse anti-human β-actin Abs (C4, Santa Cruz Biotechnology, Santa Cruz, CA) and detected via chemiluminescence using the Amersham ECL Prime Western blotting Detection System (GE Healthcare Life Sciences) and Invitrogen iBright Imaging Systems 1500 (Invitrogen, Thermo Fisher Scientific). Class II transactivator (CIITA) expression was evaluated using mouse anti-CIITA Abs (sc-13556, 7–1 H, Santa Cruz Biotechnology, Santa Cruz, CA). Phosopho-MAPK (pERK1/2) and MAPK(Erk1/2) expression was assessed using rabbit anti-phasopho-p44/42 MAPK (pERK1/2) Ab (Thr202/Tyr204, Cell Signaling Technology) and rabbit anti-p44/42 MAPK(Erk1/2) Ab (137F5, Cell Signaling Technology), respectively. MAPK siRNA was assessed using Lipofectamine ® RNAiMAX Transfection Reagent (Invitrogen, Thermo Fisher Scientific) and OptiMEM I Reduced Serum Medium (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s instructions (Supplemental Figure S1). Protein expression was analyzed using ImageJ.