Anti p jnk

Cell lysates from different cell lines were prepared with RIPA buffer in the presence of protease inhibitor cocktails and Phosphatase Inhibitor Cocktail 2 and 3 (P8340, P5726, and P0044, Sigma-Aldrich, St Louis, MO, USA). Primary antibodies included the following: anti-Ki67 (Abcam Biotechnology, Inc., Abcam, Hong Kong), anti-P21, anti-caspase3, anti-P53, anti-p-JNK, anti-JNK, anti-p-P38, anti-P38, anti-p-ERK, anti-P38, and anti-β-actin (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), and anti-PPA1 (Sigma-Aldrich St Louis, MO, USA)^{34 (link)}.

The protein samples of the right kidneys were mixed with loading buffer and subjected to SDS-PAGE. The transferred membranes were subsequently blocked and incubated overnight at 4°C with the following primary antibodies: anti-cleaved caspase-3, anti-Akt, anti-p-Akt, anti-p38, anti-p-p38, anti-ERK, anti-p-JNK, anti-JNK (all from Santa Cruz, CA, USA), anti-NF-κB p65 (both from Abcam, Cambridge, UK), and anti-p-ERK (Cell Signalling Technology, Boston, USA). β-actin (Santa Cruz, CA, USA) was blotted on the same membranes and served as the control. The protein bands were detected with SuperSignal® West Dura Extended Duration Substrate (Pierce, USA) and X-ray Film (Kodak, USA) and were then analysed with Bandscan 5.0 software and compared with β-actin.

Mouse embryos were harvested from timed pregnant females. Skulls of collected embryos were fixed in 4% Paraformaldehyde at 4°C for 24 h, then decalcified with formic acid for 14 days. Heads were sectioned (7 μm thickness) and stained with hematoxylin and eosin (H&E) or Azan. Immunofluorescence was performed in PBS/10% bovine serum albumin using the following antibodies: anti-Runx2 (Abcam,ab76956), anti-Sox9 (Abcam, ab3697; 1:50), anti-Ki67 (BD, 550609; 1:50), anti-Sp7/Osterix (Abcam, ab22552; 1:100), anti-Caspase3 (Bioss, bs-0081R), anti-GFP (Abcam, ab13970), anti-COLX (Abcam, ab58632), anti-COLII (Abcam, ab34712), anti-Caspase3 (Abcam, ab44976), anti-Scleraxis (Santa Cruz, sc-518082), Anti-FGF18 (Santa Cruz, sc-393471), anti-Collagen Type I (Proteintech, 14695-1-AP), anti-IHH (Proteintech, 13388-1-AP), anti-β-catenin (CST, #8480), anti-pErk1/2 (CST, #4370), anti-pP38 (CST, #4511), anti-pJNK (Santa Cruz, sc-6254). For immunofluorescence staining, an appropriate secondary antibody conjugated to a fluorescence probe was added. This was then incubated at room temperature for 1 h, followed by rinsing in PBS. Finally, the samples were mounted in an anti-fading mounting media. Results were obtained using an Olympus BX51 upright microscope (Olympus Optical, Tokyo, Japan).

Control and treated cells were collected and lysated as previously described and the protein amount were evaluated [43 (link), 44 (link)]. 30 μg of proteins were loaded and separated on precast 4–20% gradient Bis-Tris gel in running buffer at 100 mV for 70 min followed by transfer to PVDF membranes using a semi-dry device (Thermo scientific, UK), then blocked in 5% no-fat milk for 30 minutes. Membranes were incubated with the subsequent primary antibodies overnight: anti-p-AKT (1:1000), anti-PI3K (1:1000 Cell Signaling, USA), anti p-CREB (1:500 Cell Signaling, USA), anti-pTrkB (1:2000 Cell Signaling, USA), anti-mBDNF (1:500 Abcam, UK), anti-pJNK (1:1000 Santa Cruz, USA), anti-p75(1:1000 Abcam, UK), anti-pro-BDNF(1:1000 Millipore, USA), anti-pERK5 (1:1000 Cell Signaling, USA), anti p-ERK1,2 (1:1000 Santa Cruz, USA). After different washes, membranes were incubated with 1:10000 horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG. The protein bands were detected, normalized and analyzed to actin (housekeeping). Anti-β-actin (HRP-conjugate) (1:10000) has been used. To reprobe, membranes have been stripped with Restore stripping buffer (Thermo Scientific, UK) following manufacturer’s instructions.

Poly (lactide-co-glycolide) (PLGA)–PEG, sodium deoxycholate, Aβ_1–42 peptide and dichloromethane (DCM) were purchased from Sigma. All other chemicals used were of analytical grades. The anti-Bax, anti-Bcl-2, anti-APP, anti-BACE-1, anti-Nrf2, anti-HO-1, anti-p-NF-kB 65, anti-p-JNK, anti-TNFα, anti-iNOS, anti-p-P38, antibodies were purchased from Santa Cruz Biotechnology. Anti-caspase-3 and anti-actin antibodies were bought from Cell Signaling and anti-8-oxoguanine (anti-8-Oxo-G) were purchased from (Millipore, Billerica, MA, USA). The secondary antibodies used in our experiments were goat anti-mouse IgG, goat anti-rabbit IgG and rabbit anti-goat IgG, purchased from Santa Cruz Biotechnology.

Licochalcone A (LicA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies were used in this research: anti-Bcl-2, anti-p-JNK, anti-JNK, anti-p-ERK1/2, anti-ERK1/2, anti-p-p38, anti-p38, anti-p-Akt, anti-Akt, and anti-β-actin were purchased from Santa Cruz (CA, USA). siRNA-Beclin1, siRNA-Akt, siRNA-Atg12 were purchased from Santa Cruz (CA, USA). The anti-cleaved-caspase-3, anti-cleaved-caspase-9, and anti-cleaved-PARP, mTOR Pathway and Autophagy Antibody Sampler Kit were purchased from Cell Signal Technology. Acidic vesicular organelle was purchased from AAT Bioquest, Inc. Annexin V-FITC and propidiumiodide (PI) kit was purchased from BD Biosciences (San Diego, CA). Z-VAD-FMK was purchased from BioVision. Bafilomycin A1 was purchased from Enzo Life Sciences. LY294002 and Rapmycin were purchased from Calbiochem. pBABEpuro GFP-LC3 was a gift from Jayanta Debnath (Addgene plasmid # 22405).