p hsp 27  (Cell Signaling Technology Inc)

Journal: Biology Open

Article Title: Analysis of potential TAK1/Map3k7 phosphorylation targets in hypertrophy and cachexia models of skeletal muscle

doi: 10.1242/bio.060487

Figure Lengend Snippet: TAK1 signaling pathways in Texel sheep muscle. (A) Western blots of p-p38, p38, p-CREB, CREB, p-HSP27, HSP27, p-JNK, and JNK, p-NLK, NLK, p-p90RSK, p90RSK, p-TAK1, TAK1, p-AKT, p-GSK3B, and p-mTOR in WT and Texel sheep. White lines mark where an empty lane was removed from the images of some blots. (B) Quantitative analysis of p-p38, p38, p-CREB, CREB, p-HSP27, HSP27, p-JNK, and JNK, p-NLK, NLK, p-p90RSK, p90RSK, p-TAK1, TAK1, p-AKT, p-GSK3B, and p-mTOR in WT and Texel sheep. (Pixel density normalized to GAPDH). Texel sheep are GS1L1 LM, GS8 LM, G19L1 LM. *** P <0.001, * P <0.05.

Article Snippet: Antibodies against the following proteins were used: Nemo-like kinase (NLK) (Cell Signaling Technology #94350, 1:1000), phospho-NLK (p-NLK) (biorbyt #orb157946 1:1000), c-Jun N-terminal kinase (JNK) (Cell Signaling Technology #9252, 1:1000), phospho-JNK p-SAPK/JNK (Cell Signaling Technology #4671, 1:1000), p90/Ribosomal s6 kinase (p90RSK) (Cell Signaling Technology #9355T, 1:1000), phospho-RSK (p-p90RSK) (Cell Signaling Technology #9341, 1:1000), p38 (Cell Signaling Technology #9212, 1:1000), phospho-p38 (p-p38) (Cell Signaling Technology #9211T, 1:1000), Heat shock protein 27 (HSP27) (NOVUS #NBP1-75477SS, 1:500), phospho-HSP27 (p-HSP27) (Invitrogen #MA5-37394, 1:1000), cAMP response element binding protein (CREB) (Cell Signaling Technology #9197, 1:1000), phospho-CREB (p-CREB) (Cell Signaling Technology #9198, 1:1000), TAK1 (Sigma-Aldrich, #AB1305414, 1:1000), phospho-TAK1 (p-TAK1) (Invitrogen #MA5-15073, 1:10,000, phospho-glycogen synthase kinase 3b (GSK3B) (Cell Signaling Technology #9323T), 1:1000, phospho-mammalian target of rapamycin (p-mTOR) (Cell Signaling Technology #5536T, 1:1000, vinculin (Abcam #ab130007), myogenic factor 5 (MYF5) (R&D Systems), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [conjugated with horse radish peroxidase (HRP) (Invitrogen, #MA5-31457, 1:5000)].

Techniques: Western Blot

Journal: Biology Open

Article Title: Analysis of potential TAK1/Map3k7 phosphorylation targets in hypertrophy and cachexia models of skeletal muscle

doi: 10.1242/bio.060487

Figure Lengend Snippet: Western blot analysis of Cachexia samples. (A) Western blot of p38, p-p38, JNK, p-JNK, p90RSK, p-p90RSK, TAK1, p-TAK1, HSP27, p-hsp27. (B) Quantitative analysis of p38, p-p38, JNK, p-JNK, p90RSK, p-p90RSK, TAK1, p-TAK1, HSP27, and p-HSP27. (Pixel density normalized to GAPDH). ** P <0.01, * P <0.05.

Article Snippet: Antibodies against the following proteins were used: Nemo-like kinase (NLK) (Cell Signaling Technology #94350, 1:1000), phospho-NLK (p-NLK) (biorbyt #orb157946 1:1000), c-Jun N-terminal kinase (JNK) (Cell Signaling Technology #9252, 1:1000), phospho-JNK p-SAPK/JNK (Cell Signaling Technology #4671, 1:1000), p90/Ribosomal s6 kinase (p90RSK) (Cell Signaling Technology #9355T, 1:1000), phospho-RSK (p-p90RSK) (Cell Signaling Technology #9341, 1:1000), p38 (Cell Signaling Technology #9212, 1:1000), phospho-p38 (p-p38) (Cell Signaling Technology #9211T, 1:1000), Heat shock protein 27 (HSP27) (NOVUS #NBP1-75477SS, 1:500), phospho-HSP27 (p-HSP27) (Invitrogen #MA5-37394, 1:1000), cAMP response element binding protein (CREB) (Cell Signaling Technology #9197, 1:1000), phospho-CREB (p-CREB) (Cell Signaling Technology #9198, 1:1000), TAK1 (Sigma-Aldrich, #AB1305414, 1:1000), phospho-TAK1 (p-TAK1) (Invitrogen #MA5-15073, 1:10,000, phospho-glycogen synthase kinase 3b (GSK3B) (Cell Signaling Technology #9323T), 1:1000, phospho-mammalian target of rapamycin (p-mTOR) (Cell Signaling Technology #5536T, 1:1000, vinculin (Abcam #ab130007), myogenic factor 5 (MYF5) (R&D Systems), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [conjugated with horse radish peroxidase (HRP) (Invitrogen, #MA5-31457, 1:5000)].

Techniques: Western Blot

Journal: Biology Open

Article Title: Analysis of potential TAK1/Map3k7 phosphorylation targets in hypertrophy and cachexia models of skeletal muscle

doi: 10.1242/bio.060487

Figure Lengend Snippet: TAK1 signaling pathways in differentiated C2C12 cells. (A) Myotube formation after plating C2C12 cells at day 0 and day 8. (B) Western blots of p-TAK1, TAK1, p-p38, p38, p-NLK, NLK, p-p90RSK, p90RSK, p-HSP27, HSP27, p-JNK, and JNK in C2C12 cells at day 0 and day 8. (C) Quantitative analysis of TAK1, p-TAK1, NLK, p-NLK, p38, p-p38, HSP27, p-HSP27, p90RSK, p-p90RSK, JNK, and p-JNK2. (Pixel density normalized to GAPDH). Scale bars: 50 μm. **** P <0.0001, *** P <0.001, ** P <0.01, * P <0.05 significance.

Article Snippet: Antibodies against the following proteins were used: Nemo-like kinase (NLK) (Cell Signaling Technology #94350, 1:1000), phospho-NLK (p-NLK) (biorbyt #orb157946 1:1000), c-Jun N-terminal kinase (JNK) (Cell Signaling Technology #9252, 1:1000), phospho-JNK p-SAPK/JNK (Cell Signaling Technology #4671, 1:1000), p90/Ribosomal s6 kinase (p90RSK) (Cell Signaling Technology #9355T, 1:1000), phospho-RSK (p-p90RSK) (Cell Signaling Technology #9341, 1:1000), p38 (Cell Signaling Technology #9212, 1:1000), phospho-p38 (p-p38) (Cell Signaling Technology #9211T, 1:1000), Heat shock protein 27 (HSP27) (NOVUS #NBP1-75477SS, 1:500), phospho-HSP27 (p-HSP27) (Invitrogen #MA5-37394, 1:1000), cAMP response element binding protein (CREB) (Cell Signaling Technology #9197, 1:1000), phospho-CREB (p-CREB) (Cell Signaling Technology #9198, 1:1000), TAK1 (Sigma-Aldrich, #AB1305414, 1:1000), phospho-TAK1 (p-TAK1) (Invitrogen #MA5-15073, 1:10,000, phospho-glycogen synthase kinase 3b (GSK3B) (Cell Signaling Technology #9323T), 1:1000, phospho-mammalian target of rapamycin (p-mTOR) (Cell Signaling Technology #5536T, 1:1000, vinculin (Abcam #ab130007), myogenic factor 5 (MYF5) (R&D Systems), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [conjugated with horse radish peroxidase (HRP) (Invitrogen, #MA5-31457, 1:5000)].

Techniques: Western Blot

Journal: Biology Open

Article Title: Analysis of potential TAK1/Map3k7 phosphorylation targets in hypertrophy and cachexia models of skeletal muscle

doi: 10.1242/bio.060487

Figure Lengend Snippet: Western blot analysis of C2C12 cells on 16 h after OXO treatment. (A) Western blots of HSP27, p-HSP27, NLK, p-NLK, p90RSK, p-p90RSK, p38, p-p38, and JNK, p-JNK. (B) Quantitative analysis of p38, p-p38, JNK, p-JNK, p90RSK, p-p90RSK, NLK, p-NLK, HSP27, and p-HSP27. (Pixel density normalized to GAPDH). *** P <0.001, ** P <0.01, * P <0.05.

Article Snippet: Antibodies against the following proteins were used: Nemo-like kinase (NLK) (Cell Signaling Technology #94350, 1:1000), phospho-NLK (p-NLK) (biorbyt #orb157946 1:1000), c-Jun N-terminal kinase (JNK) (Cell Signaling Technology #9252, 1:1000), phospho-JNK p-SAPK/JNK (Cell Signaling Technology #4671, 1:1000), p90/Ribosomal s6 kinase (p90RSK) (Cell Signaling Technology #9355T, 1:1000), phospho-RSK (p-p90RSK) (Cell Signaling Technology #9341, 1:1000), p38 (Cell Signaling Technology #9212, 1:1000), phospho-p38 (p-p38) (Cell Signaling Technology #9211T, 1:1000), Heat shock protein 27 (HSP27) (NOVUS #NBP1-75477SS, 1:500), phospho-HSP27 (p-HSP27) (Invitrogen #MA5-37394, 1:1000), cAMP response element binding protein (CREB) (Cell Signaling Technology #9197, 1:1000), phospho-CREB (p-CREB) (Cell Signaling Technology #9198, 1:1000), TAK1 (Sigma-Aldrich, #AB1305414, 1:1000), phospho-TAK1 (p-TAK1) (Invitrogen #MA5-15073, 1:10,000, phospho-glycogen synthase kinase 3b (GSK3B) (Cell Signaling Technology #9323T), 1:1000, phospho-mammalian target of rapamycin (p-mTOR) (Cell Signaling Technology #5536T, 1:1000, vinculin (Abcam #ab130007), myogenic factor 5 (MYF5) (R&D Systems), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [conjugated with horse radish peroxidase (HRP) (Invitrogen, #MA5-31457, 1:5000)].

Techniques: Western Blot

Journal: FEBS open bio

Article Title: Bag-1-mediated HSF1 phosphorylation regulates expression of heat shock proteins in breast cancer cells.

doi: 10.1002/2211-5463.13843

Figure Lengend Snippet: Fig. 2. Expression of Bag-1 increases the expression of heat shock proteins by affecting HSF1. Immunoblot analysis of total heat shock protein levels in cell lysates from Bag-1 overexpressed, Bag-1-silenced and untransfected (A) MCF-7 and (B) BT-474 cells. Total protein was analyzed by immunoblotting with specific antibodies against HSF-1, phospho-HSF1Ser32 6, HSP90, HSP70, HSP27 and phospho-HSPSer78. The all data are presented as the mean SD from three biological independent experiments. Expression levels were analyzed with a t-test after normalization to vinculin. The t-test was used to calculate P values (*P < 0.05; ***P < 0.001 and ****P < 0.0001).

Article Snippet: Bag-1 (dilution 1 : 500, cat. no. #3920; Cell Signaling Technology, Inc., Beverly, MA, USA), vinculin (dilution 1 : 500, cat. no. #13901; Cell Signaling Technology, Inc.), HER2 (dilution 1 : 500, cat. no. #2165; Cell Signaling Technology, Inc.), AKT (dilution 1 : 500, cat. no. #4685; Cell Signaling Technology, Inc.), phosphorylated (p)-AKT (Ser473) (dilution 1 : 500, cat. no. #4060; Cell Signaling Technology, Inc.), PI3K (dilution 1 : 500, cat. no. #4257; Cell Signaling Technology, Inc.), mTOR (dilution 1 : 500, cat. no. #2983; Cell Signaling Technology, Inc.), p-mTOR (Ser2448) (dilution 1 : 500, cat. no. #2971; Cell Signaling Technology, Inc.), HSF1 (dilution 1 : 500, cat. no. #4356; Cell Signaling Technology, Inc.), p-HSF1 (Ser326) (dilution 1 : 500, cat. no. #ab115702; Abcam, Cambridge, UK), HSP90 (dilution 1 : 500, cat. no. #4877; Cell Signaling Technology, Inc.), HSP70 (dilution 1 : 500, cat. no. #4873; Cell Signaling Technology, Inc.), HSP27 (dilution 1 : 500, cat. no. #95357; Cell Signaling Technology, Inc.) and p-HSP27 (Ser78) (dilution 1 : 500, cat. no. #2405; Cell Signaling Technology, Inc.) antibodies were used.

Techniques: Expressing, Western Blot

Journal: FEBS open bio

Article Title: Bag-1-mediated HSF1 phosphorylation regulates expression of heat shock proteins in breast cancer cells.

doi: 10.1002/2211-5463.13843

Figure Lengend Snippet: Fig. 4. Bag-1 interacts with HSP70 and HSP27 in BT-474 cells. (A) Immunoprecipitated (IP) BT-474 cell lysates with anti-Bag-1, anti-HSP70 and anti-HSP27 antibodies were analyzed by immunoblots with Bag-1, HSP70, HSP27 and HSF1 antibodies. (B) STRING predictions for the complexes between Bag-1 and its interaction partners, and models for their cell stress response mechanisms (HSP90AA1: HSP90, HSPA4: HSP70, HSPB1: HSP27). (C) Structural predictions for the surfaces of Bag-1 (BAG domain, PDB ID: 1HXB), HSF1 (PDB ID: 5HDG), HSP90 (PDB ID: 2YI5:A, 1UYM:B), HSP70 (PDB ID: NBD: 3A8Y, SBD:4PO2) and HSP27 (PDB ID: 4MJH) proteins using crystal structures in the PDB database were analyzed by the PRISM algorithm. Interaction of the BAG domain of Bag-1 (turquoise) with the nucleotide binding and substrate binding domain of HSP70 (pink), the HSP90 alfa protein (magenta), the HSP90 beta protein (purple) and the HSP27 protein (blue). BES, binding energy score.

Article Snippet: Bag-1 (dilution 1 : 500, cat. no. #3920; Cell Signaling Technology, Inc., Beverly, MA, USA), vinculin (dilution 1 : 500, cat. no. #13901; Cell Signaling Technology, Inc.), HER2 (dilution 1 : 500, cat. no. #2165; Cell Signaling Technology, Inc.), AKT (dilution 1 : 500, cat. no. #4685; Cell Signaling Technology, Inc.), phosphorylated (p)-AKT (Ser473) (dilution 1 : 500, cat. no. #4060; Cell Signaling Technology, Inc.), PI3K (dilution 1 : 500, cat. no. #4257; Cell Signaling Technology, Inc.), mTOR (dilution 1 : 500, cat. no. #2983; Cell Signaling Technology, Inc.), p-mTOR (Ser2448) (dilution 1 : 500, cat. no. #2971; Cell Signaling Technology, Inc.), HSF1 (dilution 1 : 500, cat. no. #4356; Cell Signaling Technology, Inc.), p-HSF1 (Ser326) (dilution 1 : 500, cat. no. #ab115702; Abcam, Cambridge, UK), HSP90 (dilution 1 : 500, cat. no. #4877; Cell Signaling Technology, Inc.), HSP70 (dilution 1 : 500, cat. no. #4873; Cell Signaling Technology, Inc.), HSP27 (dilution 1 : 500, cat. no. #95357; Cell Signaling Technology, Inc.) and p-HSP27 (Ser78) (dilution 1 : 500, cat. no. #2405; Cell Signaling Technology, Inc.) antibodies were used.

Techniques: Immunoprecipitation, Western Blot, Binding Assay