Anti mkp 1

Western blots were performed on WT and AT cell protein extracts, essentially as described in [25 (link)]. After 24-h treatment, cells were collected, washed, and lysed in denaturing lysis buffer for total protein extracts. For cytosolic/nuclear protein extracts, cells were subjected to hypertonic lysis to obtain the cytosolic fraction and subsequent denaturing lysis on the nuclear pellet. Protein extracts were quantified, loaded onto the SDS-PAGE, transferred onto the nitrocellulose membrane and then subjected to immunoblotting. The following antibodies from Cell Signalling Technologies were used in the immunoblotting analyses: Phospho-AKT (Ser473), AKT, Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), p44/42 MAPK (ERK1/2), Phospho-GSK-3α/β (Ser21/9), GSK-3α/β, Fyn, LAMIN A/C, p38 MAPK, Phospho-p38 MAPK (Thr180/Tyr182) and KEAP1. Anti-MKP1, NRF2, HPRT1 and β-ACTIN were obtained from Santa Cruz Biotechnology as reported in [25 (link)]. Imaging and quantification were performed using a ChemiDoc MP Analyzer and image lab software (Bio-rad).

APP and its CTFs were detected by a polyclonal antibody C20 (1:1000) that was obtained from the laboratory of professor Weihong Song.^{49 (link)} Anti-MKP-1 (1:200, #sc-2857) was obtained from Santa Cruz Biotechnology. Anti-JNK (1:1000, #9252), anti-P-JNK (1:1000, #4668), anti-P38 (1:1000, #8690), anti-P-P38 (1:1000, #4511), anti-ERK (1:1000, #4695), anti-P-ERK (1:1000, #4370) and anti-BACE1 (1:1000, #5606) were purchased from CST. Anti-β-actin (1:3000, #A5411) antibody was purchased from Sigma.

The phosphorylation of ERK1/2 and JNK, the activation of caspase‐3 and the expression of RANKL, OPG, sclerostin, GSTP1‐1, and MKP‐1 were performed by western blot in MLO‐4Y treated as above reported. Cells were lysed in ice cold RIPA buffer containing phosphatase and protease inhibitor cocktails (Sigma) and centrifuged at 11 600 g for 10 min. Equal amounts of total proteins (40–60 μg) from whole‐cell extract were subjected under reducing conditions to SDS/PAGE on 10% gel and electrotransferred to PVDF membrane (GE Healthcare). Proteins were visualized by incubating the membranes with specific primary antibodies: anti‐caspase 3 or anti‐phospho‐ERK 1/2 or anti‐phospho‐JNK (Cell Signalling Technology, Beverly, MA, USA), or anti‐RANKL or anti‐OPG or anti‐sclerostin or anti‐GSTP1‐1 or anti‐MKP‐1 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Subsequently, membranes were stripped and reprobed with anti‐ERK1/2 or anti‐JNK or anti‐β‐actin for normalization and densitometric analysis. Secondary antibodies conjugated to horseradish peroxidase were used to detect antigen–antibody complexes with a chemiluminescence reagent kit (Bio‐Rad, Hercules, CA, USA). image j software (National Institutes of Health, Bethesda, MD, USA) was used to perform quantitative analyses, and band values were expressed as percentage relative to values of control.