Gapdh 2118

WA was purchased from Alta Vista Phytochemicals (Hyderabad, India), and biotinylation of WA was performed by Dr. P. Van der Veken (WA-BT; Universiteit Antwerpen, Belgium). Both formulations were stored as 20 mM stocks in DMSO at −20 °C as previously described [48 (link),107 (link)]. IBR (IMBRUVICA^®) stock solutions were obtained from Pharmacyclics (Sunnyvale, CA, USA). Antibodies BTK (3533) and GAPDH (2118S) were obtained from Cell Signaling Technology (Danvers, MA, USA).

HUVECs were lysed in radioimmunoprecipitation assay lysis buffer (purchased from Solarbio, China). Protein samples (20 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, China), which was then incubated with different primary antibodies, including CD31 (ab9498), vimentin (ab92547), DUSP22 (ab70124), α-SMA (ab124964), E-cadherin (ab40772), VE-cadherin (ab33168), smad2 (ab40855), smad3 (ab40854), smad2 (phospho S467) (ab280888), smad3 (phosphoS423+S425) (ab52903), ERK1+ERK2 (ab184699), ERK1 (phospho T202)+ERK2 (phospho T185) (ab201015), JNK1+JNK2+JNK3 (ab179461), and JNK1+JNK2+JNK3 (phosphoT183+T183+T221) (ab124956) (purchased from Abcam, USA) and GAPDH (2118S) and N-cadherin (13116S) (purchased from Cell Signaling Technology, USA). The blots were developed with enhanced chemiluminescence reagents (purchased from Millipore, USA) and captured by chemiluminescence imager (AI680). GAPDH served as an internal reference protein.

Antibodies against TIMP3 (5673), phospho (p)-STAT3 (9145S), STAT3 (12640S), p-Akt (Akt kinase) (4060S), t (total)-Akt (4685S), B-Cell CLL/lymphoma 2 (Bcl-2) (4223S), Bcl-2 associated X protein (Bax) (5023S), cleaved Caspase-3 (1050S), Caspase-3 (14220S), cleaved caspase-9 (20750S), caspase-9 (9502S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118S), and secondary antibodies (5151S) were purchased from Cell Signaling Technology, Inc. (CST, Danvers, MA, USA). Cisplatin was purchased from Sigma-Aldrich, Inc. (Darmstadt, Germany, P4394). IL-6 was bought from PeproTech., Inc. (Rocky Hill, NJ, United States, 200-06).

Monocytic/macrophage cell line (RAW 264.7) was obtained from American Type Culture Collection (ATCC^® TIB‐71). Soluble (s) RANKL (315‐11‐100 UG) and M‐CSF (315‐02‐100 UG) were obtained from PeproTech Incorporation. The tartrate‐resistant acid phosphatase (TRAP) assay kit (387A‐1KT) was obtained from Sigma‐Aldrich Corporation. DAPI (D1306) was purchased from Invitrogen Corporation. Antibodies for NFAT2/NFATc1 (8032) and GAPDH (2118S) were purchased from Cell Signaling Technology. Antibody for osteoprotegerin (sc‐390518) was purchased from Santa Cruz Biotechnology. The cathepsin K (ab188604), TRAP (ab185716) and MMP9 (ab38898) antibodies were purchased from Abcam.

The protein supernatant was extracted from intestinal tissues. Protein concentration was determined using the BCA method, supplemented with PBS and loading buffer to adjust the final protein concentration of the sample to 2 µg/µL, and boiled in boiling water for 10 min. and finally quickly put the sample on ice, moved to − 80 °C refrigerator until used. SDS-PAGE was used to separate the protein samples (10 µL sample per lane). The steps of electrophoresis, membrane transfer, closure and antibody incubation are performed according to the previous methods in our lab^{20 (link)}. The following antibodies were used in this study: antibodies against PI3 Kinase p85 (4292), phospho-PI3 Kinase (Tyr458, 4228S), Akt (9272S), phospho-Akt (Ser473, 9271S) and GAPDH (2118S) were all purchased from Cell Signaling Technology. On account of mammalian antibodies being used, amino acid sequences of studied protein from hybrid grouper were aligned in the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to check the identity of the antibodies. The western bands were quantified using NIH Image 1.63 software.

BGB‐283, compound C, and pimasertib were synthesized in‐house and exceeded a purity of 99% as measured by proton nuclear magnetic resonance (HNMR), liquid chromatography–mass spectrometry (LC‐MS), and high‐performance liquid chromatography (HPLC). Compounds were purchased from following source: vemurafenib, WuXi AppTec (Shanghai, China); selumetinib, PD‐0325901, and trametinib, BioChemPartner (Shanghai, China); and RO5126766, Active Biochem (Kowloon, Hong Kong). Stock solutions of compounds were prepared in dimethyl sulfoxide. Antibodies used were obtained commercially from the following sources: anti‐B‐RAF (SC‐5248), Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti‐C‐RAF (610152), BD Biosciences (San Jose, CA, USA); antibodies to MEK (9122), MEK1 (2352), phospho‐MEK1/2 (Ser217/221) (9154), ERK (4695), phospho‐ERK1/2 (Thr202/Tyr204) (4370), GAPDH (2118s), and anti‐rabbit IgG horseradish peroxidase (HRP)‐linked secondary antibody, Cell Signaling Technology (Danvers, MA, USA); and anti‐mouse IgG HRP‐linked secondary antibody (A0168), Sigma‐Aldrich (St. Louis, MO, USA). BALB/c nude mice (female) were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China). All procedures involving animals were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) of BeiGene.