Blocking Assay:Article Title: High resolution profiling of cell cycle-dependent protein and phosphorylation abundance changes in non-transformed cells
Article Snippet: .. After 1 h blocking with 5% milk in TBST at room temperature (RT) for 1 h, membranes were incubated overnight at 4 °C with the following primary antibodies: CCND1 (Santa Cruz Biotechnology, sc-20044, 1:500), CCND3 (Santa Cruz Biotechnology, sc-6283, 1:500), CCNE2 (Santa Cruz Biotechnology, sc-248, 1:500), CCNA (Santa Cruz Biotechnology, sc-271682, 1:500), CCNA (Santa Cruz Biotechnology, sc-596, 1:500), CCNB1 (Santa Cruz Biotechnology, sc-245, 1:500), GAPDH (Santa Cruz Biotechnology, sc-51907, 1:500), CDK1 pT161 (Cell Signalling Technology, 9114, 1:1000), CDK1 pY15 (Cell Signalling Technology, 9111, 1:1000), Alpha Tubulin (Abcam, ab52866, 1:5000), PLK1(Millipore, 05-844, 1:1000), CDK2 (Santa Cruz Biotechnology, sc-163, 1:500), CDK1 (Abcam, ab32094, 1:1000), CDC20 (Santa Cruz Biotechnology, sc-13162, 1:500), UBR7 (Thermo Fisher Scientific, A304-130A, 1:1000), UBE2S (Cell Signalling Technology, 11878, 1:1000), CDKN1B (Dako, M7203, 1:1000), CDKN1B pS10 (Santa Cruz Biotechnology, sc-12939, 1:500), NCL (Abcam, ab22758, 1:2000), NCL pT76 (Abcam, ab168363, 1:1000), CCNB1 (Santa Cruz Biotechnology, sc-245, 1:500), SGO2 (Thermo Fisher Scientific, A301-261A, 1:1000), GTSE1 (Abnova, H00051512-B01P, 1:1000), MELK (Thermo Fisher Scientific, A303-136A, 1:1000), FAM83D (Custom antibody from Erich Nigg, 1:500), ANAPC4 (Thermo Fisher Scientific, A301-175A, 1:1000), Phospho-CDK substrate motif [(K/H)pSP] (Cell Signalling Technology, 9477, 1:1000), JUNB (Proteintech, 10486-1-AP, 1:1000), CHAF1B (Thermo Fisher Scientific, A301-085A, 1:2000), Histone H3 pS10 (Cell Signalling Technology, 3377, 1:1000), Actin (Sigma-Aldrich, A5441, 1:5000). .. After washed with TBST for three times, membranes were incubated for 1 h at RT with the following secondary antibodies: IRDye 680RD Donkey anti-Rabbit IgG (H + L) (LI-COR Biosciences, 926-68073, 1:10000), IRDye 800CW Donkey anti-Rabbit IgG (H + L) (LI-COR Biosciences, 926-32213, 1:10000), IRDye 800CW Donkey anti-Mouse IgG (H + L) (LI-COR Biosciences, 926-32212, 1:10000), IRDye 680RD Donkey anti-Rabbit IgG (H + L) (LI-COR Biosciences, 926-68073, 1:10000), anti-mouse HRP (Cell Signalling Technology, 7076, 1:2000), anti-rabbit HRP (Cell Signalling Technology, 7074, 1:2000).
Article Title: High resolution profiling of cell cycle-dependent protein and phosphorylation abundance changes in non-transformed cells.
Article Snippet: .. After 1 h blocking with 5% milk in TBST at room temperature (RT) for 1 h, membranes were incubated overnight at 4 °C with the following primary antibodies: CCND1 (Santa Cruz Biotechnology, sc-20044, 1:500), CCND3 (Santa Cruz Biotechnology, sc-6283, 1:500), CCNE2 (Santa Cruz Biotechnology, sc-248, 1:500), CCNA (Santa Cruz Biotechnology, sc-271682, 1:500), CCNA (Santa Cruz Biotechnology, sc-596, 1:500), CCNB1 (Santa Cruz Biotechnology, sc-245, 1:500), GAPDH (Santa Cruz Biotechnology, sc-51907, 1:500), CDK1 pT161 (Cell Signalling Technology, 9114, 1:1000), CDK1 pY15 (Cell Signalling Technology, 9111, 1:1000), Alpha Tubulin (Abcam, ab52866, 1:5000), PLK1(Millipore, 05-844, 1:1000), CDK2 (Santa Cruz Biotechnology, sc-163, 1:500), CDK1 (Abcam, ab32094, 1:1000), CDC20 (Santa Cruz Biotechnology, sc-13162, 1:500), UBR7 (Thermo Fisher Scientific, A304-130A, 1:1000), UBE2S (Cell Signalling Technology, 11878, 1:1000), CDKN1B (Dako, M7203, 1:1000), CDKN1B pS10 (Santa Cruz Biotechnology, sc-12939, 1:500), NCL (Abcam, ab22758, 1:2000), NCL pT76 (Abcam, ab168363, 1:1000), CCNB1 (Santa Cruz Biotechnology, sc-245, 1:500), SGO2 (Thermo Fisher Scientific,A301-261A, 1:1000), GTSE1 (Abnova, H00051512-B01P, 1:1000), MELK (Thermo Fisher Scientific, A303-136A, 1:1000), FAM83D (Custom antibody from Erich Nigg, 1:500), ANAPC4 (Thermo Fisher Scientific, A301-175A, 1:1000), Phospho-CDK substrate motif [(K/H) pSP] (Cell Signalling Technology, 9477, 1:1000), JUNB (Proteintech, 10486-1-AP, 1:1000), CHAF1B (Thermo Fisher Scientific, A301-085A, 1:2000), Histone H3 pS10 (Cell Signalling Technology, 3377, 1:1000), Actin (Sigma-Aldrich, A5441, 1:5000). .. Afterwashedwith TBST for three times, membranes were incubated for 1 h at RT with the following secondary antibodies: IRDye 680RDDonkey anti-Rabbit IgG (H + L) (LICOR Biosciences, 926-68073, 1:10000), IRDye 800CW Donkey antiRabbit IgG (H+ L) (LI-COR Biosciences, 926-32213, 1:10000), IRDye 800CW Donkey anti-Mouse IgG (H+ L) (LI-COR Biosciences, 926- 32212, 1:10000), IRDye 680RD Donkey anti-Rabbit IgG (H + L) (LI-COR Biosciences, 926-68073, 1:10000), anti-mouse HRP (Cell Signalling Technology, 7076, 1:2000), anti-rabbit HRP (Cell Signalling Technology, 7074, 1:2000).
Incubation:Article Title: High resolution profiling of cell cycle-dependent protein and phosphorylation abundance changes in non-transformed cells
Article Snippet: .. After 1 h blocking with 5% milk in TBST at room temperature (RT) for 1 h, membranes were incubated overnight at 4 °C with the following primary antibodies: CCND1 (Santa Cruz Biotechnology, sc-20044, 1:500), CCND3 (Santa Cruz Biotechnology, sc-6283, 1:500), CCNE2 (Santa Cruz Biotechnology, sc-248, 1:500), CCNA (Santa Cruz Biotechnology, sc-271682, 1:500), CCNA (Santa Cruz Biotechnology, sc-596, 1:500), CCNB1 (Santa Cruz Biotechnology, sc-245, 1:500), GAPDH (Santa Cruz Biotechnology, sc-51907, 1:500), CDK1 pT161 (Cell Signalling Technology, 9114, 1:1000), CDK1 pY15 (Cell Signalling Technology, 9111, 1:1000), Alpha Tubulin (Abcam, ab52866, 1:5000), PLK1(Millipore, 05-844, 1:1000), CDK2 (Santa Cruz Biotechnology, sc-163, 1:500), CDK1 (Abcam, ab32094, 1:1000), CDC20 (Santa Cruz Biotechnology, sc-13162, 1:500), UBR7 (Thermo Fisher Scientific, A304-130A, 1:1000), UBE2S (Cell Signalling Technology, 11878, 1:1000), CDKN1B (Dako, M7203, 1:1000), CDKN1B pS10 (Santa Cruz Biotechnology, sc-12939, 1:500), NCL (Abcam, ab22758, 1:2000), NCL pT76 (Abcam, ab168363, 1:1000), CCNB1 (Santa Cruz Biotechnology, sc-245, 1:500), SGO2 (Thermo Fisher Scientific, A301-261A, 1:1000), GTSE1 (Abnova, H00051512-B01P, 1:1000), MELK (Thermo Fisher Scientific, A303-136A, 1:1000), FAM83D (Custom antibody from Erich Nigg, 1:500), ANAPC4 (Thermo Fisher Scientific, A301-175A, 1:1000), Phospho-CDK substrate motif [(K/H)pSP] (Cell Signalling Technology, 9477, 1:1000), JUNB (Proteintech, 10486-1-AP, 1:1000), CHAF1B (Thermo Fisher Scientific, A301-085A, 1:2000), Histone H3 pS10 (Cell Signalling Technology, 3377, 1:1000), Actin (Sigma-Aldrich, A5441, 1:5000). .. After washed with TBST for three times, membranes were incubated for 1 h at RT with the following secondary antibodies: IRDye 680RD Donkey anti-Rabbit IgG (H + L) (LI-COR Biosciences, 926-68073, 1:10000), IRDye 800CW Donkey anti-Rabbit IgG (H + L) (LI-COR Biosciences, 926-32213, 1:10000), IRDye 800CW Donkey anti-Mouse IgG (H + L) (LI-COR Biosciences, 926-32212, 1:10000), IRDye 680RD Donkey anti-Rabbit IgG (H + L) (LI-COR Biosciences, 926-68073, 1:10000), anti-mouse HRP (Cell Signalling Technology, 7076, 1:2000), anti-rabbit HRP (Cell Signalling Technology, 7074, 1:2000).
Article Title: A signature of correlated CDC20 and UBCH10 expression indicates poor prognosis in primary head and neck squamous cell carcinoma
Article Snippet: Equal amounts of protein samples were separated using SDS-PAGE (8–12%), transferred to activated PVDF membranes (Amersham, GE Healthcare Life Sciences). .. The membranes were incubated with primary antibodies specific for Cdc20 (Santa Cruz Biotechnology, Inc.), UbcH10 (ABclonal Science, Inc.), and beta-actin (Abcam) at 4 °C overnight according to the manufacturer’s instructions. .. Corresponding HRP-conjugated secondary antibodies (Sigma‒Aldrich) were used, and the bands were visualized with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.) in a Bio-Rad gel documentation system.
Article Title: High resolution profiling of cell cycle-dependent protein and phosphorylation abundance changes in non-transformed cells.
Article Snippet: .. After 1 h blocking with 5% milk in TBST at room temperature (RT) for 1 h, membranes were incubated overnight at 4 °C with the following primary antibodies: CCND1 (Santa Cruz Biotechnology, sc-20044, 1:500), CCND3 (Santa Cruz Biotechnology, sc-6283, 1:500), CCNE2 (Santa Cruz Biotechnology, sc-248, 1:500), CCNA (Santa Cruz Biotechnology, sc-271682, 1:500), CCNA (Santa Cruz Biotechnology, sc-596, 1:500), CCNB1 (Santa Cruz Biotechnology, sc-245, 1:500), GAPDH (Santa Cruz Biotechnology, sc-51907, 1:500), CDK1 pT161 (Cell Signalling Technology, 9114, 1:1000), CDK1 pY15 (Cell Signalling Technology, 9111, 1:1000), Alpha Tubulin (Abcam, ab52866, 1:5000), PLK1(Millipore, 05-844, 1:1000), CDK2 (Santa Cruz Biotechnology, sc-163, 1:500), CDK1 (Abcam, ab32094, 1:1000), CDC20 (Santa Cruz Biotechnology, sc-13162, 1:500), UBR7 (Thermo Fisher Scientific, A304-130A, 1:1000), UBE2S (Cell Signalling Technology, 11878, 1:1000), CDKN1B (Dako, M7203, 1:1000), CDKN1B pS10 (Santa Cruz Biotechnology, sc-12939, 1:500), NCL (Abcam, ab22758, 1:2000), NCL pT76 (Abcam, ab168363, 1:1000), CCNB1 (Santa Cruz Biotechnology, sc-245, 1:500), SGO2 (Thermo Fisher Scientific,A301-261A, 1:1000), GTSE1 (Abnova, H00051512-B01P, 1:1000), MELK (Thermo Fisher Scientific, A303-136A, 1:1000), FAM83D (Custom antibody from Erich Nigg, 1:500), ANAPC4 (Thermo Fisher Scientific, A301-175A, 1:1000), Phospho-CDK substrate motif [(K/H) pSP] (Cell Signalling Technology, 9477, 1:1000), JUNB (Proteintech, 10486-1-AP, 1:1000), CHAF1B (Thermo Fisher Scientific, A301-085A, 1:2000), Histone H3 pS10 (Cell Signalling Technology, 3377, 1:1000), Actin (Sigma-Aldrich, A5441, 1:5000). .. Afterwashedwith TBST for three times, membranes were incubated for 1 h at RT with the following secondary antibodies: IRDye 680RDDonkey anti-Rabbit IgG (H + L) (LICOR Biosciences, 926-68073, 1:10000), IRDye 800CW Donkey antiRabbit IgG (H+ L) (LI-COR Biosciences, 926-32213, 1:10000), IRDye 800CW Donkey anti-Mouse IgG (H+ L) (LI-COR Biosciences, 926- 32212, 1:10000), IRDye 680RD Donkey anti-Rabbit IgG (H + L) (LI-COR Biosciences, 926-68073, 1:10000), anti-mouse HRP (Cell Signalling Technology, 7076, 1:2000), anti-rabbit HRP (Cell Signalling Technology, 7074, 1:2000).
other:Article Title: Oncogenic and immunological roles of LAMP5 across cancers and its potential utility in bladder cancer.
Article Snippet: Zhenni Lin and Kun Zhao contributed equally to this work.. Abstract Lysosomal associated membrane protein family member 5 (LAMP5), a recently identified member of the LAMP family, has been associated with poor prognosis in multiple cancer types; however, its precise oncogenic mechanisms remain unclear.. This study systematically investigated the oncogenic and immunological functions of LAMP5 using multiple datasets.
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