A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots

Blake, M. S.; Johnston, Kenneth H.; Russell-Jones, G.J.; Gotschlich, Emil C.

doi:10.1016/0003-2697(84)90320-8

Cited by 2,107 publications

(822 citation statements)

References 14 publications

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“…Cell lysates were heated at 1008C for 5 min, and applied to 10% polyacrylamide gels. Western blotting was done by standard procedures using an alkaline phosphatase-conjugated second antibody (Blake et al, 1984).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of the growth of WI-38 fibroblasts by benzyloxycarbonyl-Leu-Leu-Tyr diazomethyl ketone: evidence that cleavage of p53 by a calpain-like protease is necessary for G1 to S-phase transition

et al. 1997

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The e ect of a calpain-selective cell permeant inhibitor, benzyloxycarbonyl Leu-Leu-Tyr diazomethylketone (ZLLY-CHN 2 ), on the serum-stimulated growth of WI-38 human ®broblasts has been investigated. Only cell permeant protease inhibitors with activity against calpains prevented progression into S-phase. Protein blotting experiments indicated that p53 immunoreactivity increased in late G 1 cells treated with ZLLY-CHN 2 . The content of p21 Waf1/Cip1 CDK inhibitor also increased, providing a mechanism for the observed failure to enter S-phase. Further studies indicated that p53 could be degraded by a ZLLY-CHN 2 -sensitive protease immediately prior to S-phase, but that proteolysis did not occur after this critical time point. Chelation of extracellular Ca 2+ by addition of EGTA inhibited the p53 degradation. Consistent with proteolysis of p53 in late G 1 phase, m-calpain immunoreactivity transiently accumulated in cell nuclei at this time. ZLLY-CHN 2 did not appear to increase p53 mRNA in WI-38 cells. Puri®ed mcalpain required only 1 to 3 mM Ca 2+ to proteolyze p53 in WI-38 cell lysates. These results indicate that ZLLY-CHN 2 inhibits progression of WI-38 cells into S-phase by inactivating a calpain-like protease that is responsible for proteolysis of constitutively expressed p53 in late G 1 .

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Section: Methodsmentioning

confidence: 99%

Inhibition of the growth of WI-38 fibroblasts by benzyloxycarbonyl-Leu-Leu-Tyr diazomethyl ketone: evidence that cleavage of p53 by a calpain-like protease is necessary for G1 to S-phase transition

et al. 1997

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“…Briefly, membranes from lys;d yeast were solubilized in Laemmli's sample buffer containing 10% S[I~S and run on a 12% polyacrylamide gel [15]. Proteins were transfel ed onto a nitrocellulose membrane using a mini trans-blot cell (BioRad) in 25 mM Tris-HC1, pH 7.4, 192 mM glycine at 150 mA for 1 hour [16]. This membrane was then blocked using phosphate-buffered saline (PBS) containing 2% BSA and 0.05% Tween 20 for 30 min.…”

Section: : Immunoblotsmentioning

confidence: 99%

The human β₂‐adrenergic receptor expressed in Schizosaccharomyces pombe retains its pharmacological properties

1995

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“…50/~g protein per lane were run on SDS gels, transferred to a nylon membrane [23], and probed with rabbit anti-human CA II followed by goat anti-rabbit IgG conjugated to alkaline phosphatase. Blots were developed with nitroblue tetrazolium and bromochloroindoyl phosphate [24]. …”

Section: Western Blotsmentioning

confidence: 99%

Carbonic anhydrase II is induced in HL‐60 cells by 1,25‐dihydroxyvitamin D₃: A model for osteoclast gene regulation

Shapiro

Venta

et al. 1989

FEBS Letters

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Carbonic anhydrase II (CA II) generates the H ÷ required for osteoelast-mediated bone resorption in humans. We have developed the human promyelocytic cell line HL-60 as a model system with which to study the osteoclast-specific expression of the CA II gene. Treatment of the cell line with 1,25-dihydroxyvitamin D 3 resulted in a dramatic de novo induction of CA II at both the protein and mRNA levels. CA II mRNA was also induced to a lesser extent by 12-O-tetradecanoyl phorbol 13-acetate. Treatment with dimethyl sulfoxide did not increase CA II mRNA. These findings indicate that the HL-60 cell line will be a useful model system to study the osteoclast-speeific expression of the CA II gene.Carbonic anhydrase II; Osteoclast; Dihydroxyvitamin D3, 1,25-; (HL-60 cell)

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A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots

Cited by 2,107 publications

References 14 publications

Inhibition of the growth of WI-38 fibroblasts by benzyloxycarbonyl-Leu-Leu-Tyr diazomethyl ketone: evidence that cleavage of p53 by a calpain-like protease is necessary for G1 to S-phase transition

Inhibition of the growth of WI-38 fibroblasts by benzyloxycarbonyl-Leu-Leu-Tyr diazomethyl ketone: evidence that cleavage of p53 by a calpain-like protease is necessary for G1 to S-phase transition

The human β₂‐adrenergic receptor expressed in Schizosaccharomyces pombe retains its pharmacological properties

Carbonic anhydrase II is induced in HL‐60 cells by 1,25‐dihydroxyvitamin D₃: A model for osteoclast gene regulation

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A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots

Cited by 2,107 publications

References 14 publications

Inhibition of the growth of WI-38 fibroblasts by benzyloxycarbonyl-Leu-Leu-Tyr diazomethyl ketone: evidence that cleavage of p53 by a calpain-like protease is necessary for G1 to S-phase transition

Inhibition of the growth of WI-38 fibroblasts by benzyloxycarbonyl-Leu-Leu-Tyr diazomethyl ketone: evidence that cleavage of p53 by a calpain-like protease is necessary for G1 to S-phase transition

The human β2‐adrenergic receptor expressed in Schizosaccharomyces pombe retains its pharmacological properties

Carbonic anhydrase II is induced in HL‐60 cells by 1,25‐dihydroxyvitamin D3: A model for osteoclast gene regulation

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The human β₂‐adrenergic receptor expressed in Schizosaccharomyces pombe retains its pharmacological properties

Carbonic anhydrase II is induced in HL‐60 cells by 1,25‐dihydroxyvitamin D₃: A model for osteoclast gene regulation