phosphorylated p p38mapk  (Cell Signaling Technology Inc)

Journal: Cells

Article Title: Mitophagy Effects of Protodioscin on Human Osteosarcoma Cells by Inhibition of p38MAPK Targeting NIX/LC3 Axis.

doi: 10.3390/cells12030395

Figure Lengend Snippet: Figure 5. The p38MAPK signaling pathway mediates the PD-induced mitophagy and apoptosis in human OS cells. (A,B) Human HOS and 143B OS cells were exposed to various concentrations (0, 2, 4, and 6 µM) of PD for 30 min. Cells were harvested and examined with immunoblotting to deter-mine the signaling pathway activation. HOS cells exposed to 4 µM PD and/or p38 siRNA (10 nM) were harvested for detecting (C) MMP changes, (D) AO staining, (E) mitophagy induction, and (F) the protein expressions of p38, cleaved caspase-3, cleaved PARP, NIX, and LC3B proteins, using immunoblotting assays. GAPDH was used as an internal loading control. ** p < 0.01, versus the control. # p < 0.05 versus the PD-only treatment (line 2). The results are the mean ± standard deviation of three experiments. Scale bar: 50 µm.

Article Snippet: Antibodies against cleaved caspase-caspase-3 (SC-56053), PINK1 (SC-518052), Parkin (SC-32282), NIX (SC-166332), BNIP3 (SC-56167), total p38MAPK (SC-7972), phosphoryl p38MAPK (SC-166182), total Akt (SC-56878), phosphoryl Akt (SC-271966), LC3 siRNA (SC-43390), NIX siRNA (SC-37453), and horseradish peroxidase-conjugated secondary antibodies (anti-mouse and anti-rabbit) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Western Blot, Activation Assay, Staining, Control, Standard Deviation

Journal: Bioengineered

Article Title: Effects of runt-related transcription factor 2 ( RUNX2 ) on the autophagy of rapamycin-treated osteoblasts

doi: 10.1080/21655979.2022.2037881

Figure Lengend Snippet: The sequences of all primers

Article Snippet: After 2 h of blockage, the membranes were incubated with anti-RUNX2 antibody (1:1000, ProteinTech Group, Inc.), anti-p38MAPK anti-body (1:1000, ProteinTech Group, Inc.), anti-phosphorylation (p)-p38MAPK antibody (1:1000, Cell Signaling Technology), anti-LC3 antibody (1:1000, Cell Signaling Technology), anti-Beclin-1 antibody (1:1000, Cell Signaling Technology), anti-p-Beclin-1 antibody (1:1000, Cell Signaling Technology), anti-p62 antibody (1:1000, ProteinTech Group, Inc.), anti-Autophagy-related protein 1 (ATG1) antibody (1:1000, Cell Signaling Technology), anti-Autophagy-related protein 5 (AGT5) antibody (1:1000, Cell Signaling Technology), and anti-GAPDH (1:5000, ProteinTech Group, Inc.) at 4 °C overnight.

Techniques: Sequencing

Journal: Bioengineered

Article Title: Effects of runt-related transcription factor 2 ( RUNX2 ) on the autophagy of rapamycin-treated osteoblasts

doi: 10.1080/21655979.2022.2037881

Figure Lengend Snippet: Selection of the optimum concentration of rapamycin. (a) Viability of MC3T3-E1 cells treated with different concentrations of rapamycin based on cell counting Kit-8. The mRNA expression levels of runt-related transcription factor 2 ( RUNX2 ) (b), p38 map kinase ( p38 MAPK ) (c), microtubule-associated protein 1 light chain 3 alpha ( LC3-II ) (d), Beclin-1 (e), and heat shock 90-like protein ( p62 ) (f). *: P < 0.05, compared with 0 nm rapamycin. **: P < 0.01, compared with 0 nm rapamycin.

Article Snippet: After 2 h of blockage, the membranes were incubated with anti-RUNX2 antibody (1:1000, ProteinTech Group, Inc.), anti-p38MAPK anti-body (1:1000, ProteinTech Group, Inc.), anti-phosphorylation (p)-p38MAPK antibody (1:1000, Cell Signaling Technology), anti-LC3 antibody (1:1000, Cell Signaling Technology), anti-Beclin-1 antibody (1:1000, Cell Signaling Technology), anti-p-Beclin-1 antibody (1:1000, Cell Signaling Technology), anti-p62 antibody (1:1000, ProteinTech Group, Inc.), anti-Autophagy-related protein 1 (ATG1) antibody (1:1000, Cell Signaling Technology), anti-Autophagy-related protein 5 (AGT5) antibody (1:1000, Cell Signaling Technology), and anti-GAPDH (1:5000, ProteinTech Group, Inc.) at 4 °C overnight.

Techniques: Selection, Concentration Assay, Cell Counting, Expressing

Journal: Bioengineered

Article Title: Effects of runt-related transcription factor 2 ( RUNX2 ) on the autophagy of rapamycin-treated osteoblasts

doi: 10.1080/21655979.2022.2037881

Figure Lengend Snippet: Rapamycin induced osteoblast MC3T3-E1 autophagy by regulating related genes. (a) The protein bands of related genes determined by Western blot. (b) Gray analysis of Beclin-1. (c) Gray analysis of microtubule-associated protein 1 light chain 3 alpha (LC3-II). (d) Gray analysis of p38 map kinase (p38 MAPK). (e) Gray analysis of heat shock 90-like protein (p62). (f) Gray analysis of runt-related transcription factor 2 (RUNX2). (g) Autophagosomes in cells treated with 0 nm, 10 nm, and 50 nm rapamycin treatment. (h) Quantitative analysis of autophagosomes. *: P < 0.05, compared with 0 nm rapamycin.

Article Snippet: After 2 h of blockage, the membranes were incubated with anti-RUNX2 antibody (1:1000, ProteinTech Group, Inc.), anti-p38MAPK anti-body (1:1000, ProteinTech Group, Inc.), anti-phosphorylation (p)-p38MAPK antibody (1:1000, Cell Signaling Technology), anti-LC3 antibody (1:1000, Cell Signaling Technology), anti-Beclin-1 antibody (1:1000, Cell Signaling Technology), anti-p-Beclin-1 antibody (1:1000, Cell Signaling Technology), anti-p62 antibody (1:1000, ProteinTech Group, Inc.), anti-Autophagy-related protein 1 (ATG1) antibody (1:1000, Cell Signaling Technology), anti-Autophagy-related protein 5 (AGT5) antibody (1:1000, Cell Signaling Technology), and anti-GAPDH (1:5000, ProteinTech Group, Inc.) at 4 °C overnight.

Techniques: Western Blot

Journal: Bioengineered

Article Title: Effects of runt-related transcription factor 2 ( RUNX2 ) on the autophagy of rapamycin-treated osteoblasts

doi: 10.1080/21655979.2022.2037881

Figure Lengend Snippet: Runt-related transcription factor 2 (RUNX2) overexpression enhanced the viability of rapamycin-treated MC3T3-E1 cells by regulating autophagy-related genes and osteoblast differentiation-related genes. (a) Viability of MC3T3-E1 cells without rapamycin treatment after transfection with si-NC and si-RUNX2. (b) Viability of rapamycin-treated MC3T3-E1 cells with RUNX2 overexpression or knockdown. *: P < 0.05, compared with the control group; # : P < 0.05, compared with si-RUNX2 group. The mRNA expression levels of p38 map kinase ( p38 MAPK ) (c), microtubule-associated protein 1 light chain 3 alpha ( LC3-II ) (d), Beclin-1 (e), heat shock 90-like protein ( p62 ) (f), alkaline phosphatase ( ALP ) (g), Sp7 transcription factor ( OSX ) (h), bone gamma-carboxyglutamate protein ( OCN ) (i) and collagen, type I, alpha 1 ( COL1A1 ) (j). *: P < 0.05, compared with the control group; # : P < 0.05, compared with pcDNA3.1(+)-RUNX2 group.

Article Snippet: After 2 h of blockage, the membranes were incubated with anti-RUNX2 antibody (1:1000, ProteinTech Group, Inc.), anti-p38MAPK anti-body (1:1000, ProteinTech Group, Inc.), anti-phosphorylation (p)-p38MAPK antibody (1:1000, Cell Signaling Technology), anti-LC3 antibody (1:1000, Cell Signaling Technology), anti-Beclin-1 antibody (1:1000, Cell Signaling Technology), anti-p-Beclin-1 antibody (1:1000, Cell Signaling Technology), anti-p62 antibody (1:1000, ProteinTech Group, Inc.), anti-Autophagy-related protein 1 (ATG1) antibody (1:1000, Cell Signaling Technology), anti-Autophagy-related protein 5 (AGT5) antibody (1:1000, Cell Signaling Technology), and anti-GAPDH (1:5000, ProteinTech Group, Inc.) at 4 °C overnight.

Techniques: Over Expression, Transfection, Knockdown, Control, Expressing

Journal: Bioengineered

Article Title: Effects of runt-related transcription factor 2 ( RUNX2 ) on the autophagy of rapamycin-treated osteoblasts

doi: 10.1080/21655979.2022.2037881

Figure Lengend Snippet: Effects of runt-related transcription factor 2 ( RUNX2 ) on the protein expression levels of autophagy-related proteins and p38 MAPK signaling pathway. (a) The protein bands of related proteins based on Western blot. The protein levels of p-p38 map kinase/ p38 map kinase (p-p38 MAPK/p38 MAPK) (b), Beclin-1 (c), p-Beclin-1 (d), microtubule-associated protein 1 light chain 3 alpha (LC3-II/LC3-I) (e), Autophagy-related protein 1 (ATG1) (f), Autophagy-related protein 5 (ATG5) (g), and heat shock 90-like protein (p62) (h). *: P < 0.05, compared with the control group; # : P < 0.05, compared with pcDNA3.1(+)-RUNX2 group.

Article Snippet: After 2 h of blockage, the membranes were incubated with anti-RUNX2 antibody (1:1000, ProteinTech Group, Inc.), anti-p38MAPK anti-body (1:1000, ProteinTech Group, Inc.), anti-phosphorylation (p)-p38MAPK antibody (1:1000, Cell Signaling Technology), anti-LC3 antibody (1:1000, Cell Signaling Technology), anti-Beclin-1 antibody (1:1000, Cell Signaling Technology), anti-p-Beclin-1 antibody (1:1000, Cell Signaling Technology), anti-p62 antibody (1:1000, ProteinTech Group, Inc.), anti-Autophagy-related protein 1 (ATG1) antibody (1:1000, Cell Signaling Technology), anti-Autophagy-related protein 5 (AGT5) antibody (1:1000, Cell Signaling Technology), and anti-GAPDH (1:5000, ProteinTech Group, Inc.) at 4 °C overnight.

Techniques: Expressing, Western Blot, Control