Western Blot:Article Title: Identification of myeloid-derived growth factor as a mechanically-induced, growth-promoting angiocrine signal for human hepatocytes
Article Snippet: For MACS anti-CD146 mouse MicroBeads (Miltenyi Biotec, 130-092-007) were used. .. For Western blotting following antibodies were used: rabbit anti-MYDGF (Proteintech, 11353-1-AP, Lot: 00064724, 1/1000), rabbit anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling, 4376, Lot: 21, 1/750 or 1/500), rabbit anti-p44/42 MAPK (Erk1/2) (137F5) (Cell Signaling, 4695, Lot: 28, 1/750 or 1/500), rabbit anti-Phospho-Stat3 (Ser727) (Cell Signaling, 9134, Lot: 21, 1/500), rabbit anti-Stat3 (D3Z2G) (Cell Signaling, 12640, Lot: 7, 1/750), rabbit anti-PhosphoAKT (Ser473) (D9E) (Cell Signaling, 4060, Lot: 27, 1/750), rabbit anti-AKT (pan) (C67E7) (Cell Signaling, 4691, Lot: 20, 1/750), rabbit anti-GAPDH (Abcam, ab9485, Lot: GR3437709-1, 1/5000 or 1/2000) and rabbit anti-" tubulin antibody (Abcam, ab6046, Lot: GR3441353-1, 1/2000), goat anti rabbit IgG, HRP-linked (Cell Signaling, 7074, Lot: 27, 1/2000). .. Antibodies were validated by the suppliers as follows: rabbit anti-phospho-Histone H3 (Ser10) (Sigma-Aldrich, 06-570): Immunohistochemistry (IHC), Western Blot (WB), Immunocytochemistry (ICC); rabbit anti-caspase-3 (Sigma-Aldrich, C8487): Immunofluorescent (IF), WB; rabbit anti-HNF4! (Cell Signaling, C11F12): WB, IHC,IF; goat anti-ICAM-1/CD54 (R&D, AF796): WB, IHC, adhesion blockade; rat anti-Ki-67 (SolA15) (Invitrogen, 14-5698-82): WB, IHC, ICC, Flow Cytomentry (Flow), Functional Assay (FN); rabbit anti-HNF4! (Abcam, ab181604): Chromatin Immuno-Cleavage/Cleavage Under Targets and Release Using Nuclease (ChIC/ CUT&RUN); chromatin immunoprecipitation (ChIP), IHC, WB, Immunoprecipitation (IP); goat anti-GFP (Sicgen, AB0020-200): WB, IF, IHC; donkey anti-rabbit Alexa Fluor 488 (Invitrogen, A-21206): IHC, ICC/IF, Flow; donkey anti-rat Alexa Fluor 488 (Invitrogen, A-21208): IHC, ICC/IF; donkey anti-goat Alexa Fluor 488 (Invitrogen, A-11055): IHC, ICC/IF, Flow; donkey anti-goat Alexa Fluor 555 (Invitrogen, A-21432): IHC, ICC/IF; donkey anti-rabbit Alexa Fluor 555 (Invitrogen, A-31572): IHC, ICC/IF, Flow; DAPI (Sigma-Aldrich, D9542): IF; rabbit anti-MYDGF (Proteintech, 11353-1-AP): WB (antibody was also validated in our laboratory by adding recombinant MYDGF protein (Novoprotein, CG64) on WB), IHC, IF, enzyme-linked immunosorbent assay (ELISA); rabbit anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling, 4376): WB, IP, IHC; rabbit anti-p44/42 MAPK (Erk1/2) (137F5) (Cell Signaling, 4695): WB, IP, IHC, IF, Flow; rabbit anti-Phospho-Stat3 (Ser727) (Cell Signaling, 9134): WB, IP, ChIP; rabbit anti-Stat3 (D3Z2G) (Cell Signaling, 12640): WB, IP, IF, Flow, ChIP; rabbit anti-Phospho-AKT (Ser473) (D9E) (Cell Signaling, 4060): WB, IP, IHC, IF, Flow; rabbit anti-AKT (pan) (C67E7) (Cell Signaling, 4691): WB, IP, IHC, IF, Flow; rabbit anti-GAPDH (Abcam, ab9485): IHC, WB, ICC/IF; rabbit anti-" tubulin antibody (Abcam, ab6046): WB, ICC/IF, IHC, IP; goat anti rabbit IgG, HRP-linked (Cell Signaling, 7074): WB.
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