Anti phospho erk1 2 4370

Total protein was extracted from fresh tissue or cultured cells by using Tissue Protein Extraction Reagent (Keygenbio, KGP250) containing protease and phosphatase inhibitors according to the manufacturer's instructions and centrifuge tube (Guangzhou Jet Bio‐Filtration Co., Ltd). Western blot analysis was performed as described previously.²³ The primary antibodies anti‐VEGF‐C (22601‐1‐AP, 1:500), anti‐Bcl‐2 (26593‐1‐AP, 1:1000), and FOXC2 (23066‐1‐AP, 1:1000) were purchased from Proteintech (Wuhan, CN); anti‐VEGFR‐3 (ab27278, 1:1000) and anti‐TBX1 (ab109313, 1:1000) were purchased from Abcam (Cambridge, UK); anti‐phospho‐AKT (9271S, 1:500), anti‐AKT (9272S, 1:1000), anti‐phospho‐ERK1/2 (4370S, 1:500), anti‐ERK1/2 (4695S, 1:1000), anti‐CaNA (2614S, 1:1000), anti‐Bax (2772S, 1:1000), anti‐VEGFR‐2 (9698, 1:500), and anti‐GAPDH (2118S, 1:1000) were obtained from Cell Signaling Technology, Inc (Danvers, MA, USA); anti‐NFATc1 (MA3‐024, 1:1000) was purchased from Invitrogen (Carlsbad, CA, USA); and anti‐connexin 43 (CX43, ARG55217, 1:1000) and anti‐VEGF‐D (ARG58713, 1:1000) were purchased from Arigo (Taiwan, CN). The anti‐rabbit or anti‐mouse horseradish peroxidase‐conjugated secondary antibodies were purchased from Sino Biological Inc. (1:2000).

Cells were lysed in RIPA buffer (Beyotime Biotech) to extract the proteins. Immunoblotting was performed with primary antibodies anti-Hsp70 (ab2787; Abcam), anti-TSG101 (ab125011; Abcam), anti-CD9 (ab92726; Abcam), anti-AKT (4685S; Cell Signaling Technology, Danvers, MA, USA), anti-phospho-AKT (4060S; Cell Signaling Technology), anti-ERK1/2 (4695S; Cell Signaling Technology), anti-phospho-ERK1/2 (4370S; Cell Signaling Technology), anti-Bcl-2 (ab196495; Abcam), anti-Bax (ab32503; Abcam), and anti-β-actin (ab8226; Abcam), according to the manufacturer's instructions. Goat anti-rabbit IgG (ab205718; Abcam) was used as the secondary antibody. The quantification of protein bands was performed using the ImageJ software.