anti gapdh  (Millipore)


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    Anti GAPDH
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    Structured Review

    Millipore anti gapdh
    Anti GAPDH

    https://www.bioz.com/result/anti gapdh/product/Millipore
    Average 99 stars, based on 3317 article reviews
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    anti gapdh - by Bioz Stars, 2020-04
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    Images

    1) Product Images from "Phosphorylation of the Human Papillomavirus Type 16 E1^E4 Protein at T57 by ERK Triggers a Structural Change That Enhances Keratin Binding and Protein Stability ▿Phosphorylation of the Human Papillomavirus Type 16 E1^E4 Protein at T57 by ERK Triggers a Structural Change That Enhances Keratin Binding and Protein Stability ▿ †"

    Article Title: Phosphorylation of the Human Papillomavirus Type 16 E1^E4 Protein at T57 by ERK Triggers a Structural Change That Enhances Keratin Binding and Protein Stability ▿Phosphorylation of the Human Papillomavirus Type 16 E1^E4 Protein at T57 by ERK Triggers a Structural Change That Enhances Keratin Binding and Protein Stability ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.02063-08

    T57 phosphorylation increases the stability and abundance of full-length 16E1^E4 in the cell. (A) His-16E1^E4 was incubated at 30°C in the presence (+) or absence (−) of ERK for 1 h and analyzed by Western blotting using antibodies to 16E1^E4 or to T57-phosphorylated 16E1^E4 (phospho T57). High- and low-level exposures of these blots are shown, demonstrating that the phosphospecific antibody did not detect unphosphorylated E1^E4 (upper panels). SiHa or PHK cells were infected with rAd16E1^E4 for 24 h, and cell lysates were analyzed by Western blotting using antibodies to 16E1^E4 or to T57-phosphorylated 16E1^E4. The antibody to T57-phosphorylated 16E1^E4 detects only the slower-migrating band, confirming that this band is the T57-phosphorylated form of 16E1^E4 (lower panels). (B) SiHa cells were infected with rAd16E1^E4 for 24 or 48 h, fixed with 5% formaldehyde, and triple stained with antibodies to total 16E1^E4 (green) and T57-phosphorylated 16E1^E4 (red) and with DAPI (4′,6-diamidino-2-phenylindole; blue). T57-phosphorylated 16E1^E4 appeared only in cells containing abundant E1^E4 (shown by arrows). Images were captured using a 20× objective. (C) SiHa cells were infected with rAd16E1^E4 for 24 h, and then the SiHa cells expressing high and low levels of E1^E4 were separated by FACS and analyzed by Western blotting with antibodies against 16E1^E4 and GAPDH (as a loading control). Fluorescence intensity correlated with protein abundance and the presence of the T57-phosphorylated (slower-migrating) E1^E4 form. The molecular masses of 16E^E4 forms (in kilodaltons) are shown to the right of the lower panel. +ve, positive; −ve, negative; FITC, fluorescein isothiocyanate. (D, top panel) SiHa cells were transfected with a 16E1^E4 WT-, T57A-, or T57D-expressing plasmid for 24 h prior to SDS-PAGE and Western blotting to reveal the different migration patterns. The molecular masses of 16E1^E4 forms (in kilodaltons) are shown to the left. (Bottom panels) SiHa cells were transfected with a 16E1^E4 WT- or T57D-expressing plasmid for 24 h before being treated with 40 μg/ml cycloheximide for 0, 2, 4, 6, 12, or 24 h prior to harvest. Equal volumes of the soluble protein fraction were analyzed by Western blotting to show the increased stability of 16E1^E4 T57D relative to those of the non-T57-phosphorylated WT 16E1^E4 and the GAPDH loading controls. The results shown are typical of results from triplicate experiments. (E) SiHa cells were cotransfected with a plasmid expressing either T57A, T57D, or WT 16E1^E4 and the GFP-expressing plasmid pXJ-GFP (as a transfection control). Cells were harvested at multiple time points posttransfection, as indicated. Total extracts were run on SDS-polyacrylamide gels and analyzed with antibodies against GFP and 16E1^E4 to show the accumulation of 16E1^E4 in the cell. The time course shows the more rapid accumulation of the T57D (phosphomimic) form than of the WT 16E1^E4 (which lacks a T57-phosphorylated form before the 24-h time point) and the 16E1^E4 T57A mutant (which lacks the T57 phosphorylation site). The results shown are typical of results from triplicate experiments. The molecular masses of 16E1^E4 forms (in kilodaltons) are shown to the right (left panel).
    Figure Legend Snippet: T57 phosphorylation increases the stability and abundance of full-length 16E1^E4 in the cell. (A) His-16E1^E4 was incubated at 30°C in the presence (+) or absence (−) of ERK for 1 h and analyzed by Western blotting using antibodies to 16E1^E4 or to T57-phosphorylated 16E1^E4 (phospho T57). High- and low-level exposures of these blots are shown, demonstrating that the phosphospecific antibody did not detect unphosphorylated E1^E4 (upper panels). SiHa or PHK cells were infected with rAd16E1^E4 for 24 h, and cell lysates were analyzed by Western blotting using antibodies to 16E1^E4 or to T57-phosphorylated 16E1^E4. The antibody to T57-phosphorylated 16E1^E4 detects only the slower-migrating band, confirming that this band is the T57-phosphorylated form of 16E1^E4 (lower panels). (B) SiHa cells were infected with rAd16E1^E4 for 24 or 48 h, fixed with 5% formaldehyde, and triple stained with antibodies to total 16E1^E4 (green) and T57-phosphorylated 16E1^E4 (red) and with DAPI (4′,6-diamidino-2-phenylindole; blue). T57-phosphorylated 16E1^E4 appeared only in cells containing abundant E1^E4 (shown by arrows). Images were captured using a 20× objective. (C) SiHa cells were infected with rAd16E1^E4 for 24 h, and then the SiHa cells expressing high and low levels of E1^E4 were separated by FACS and analyzed by Western blotting with antibodies against 16E1^E4 and GAPDH (as a loading control). Fluorescence intensity correlated with protein abundance and the presence of the T57-phosphorylated (slower-migrating) E1^E4 form. The molecular masses of 16E^E4 forms (in kilodaltons) are shown to the right of the lower panel. +ve, positive; −ve, negative; FITC, fluorescein isothiocyanate. (D, top panel) SiHa cells were transfected with a 16E1^E4 WT-, T57A-, or T57D-expressing plasmid for 24 h prior to SDS-PAGE and Western blotting to reveal the different migration patterns. The molecular masses of 16E1^E4 forms (in kilodaltons) are shown to the left. (Bottom panels) SiHa cells were transfected with a 16E1^E4 WT- or T57D-expressing plasmid for 24 h before being treated with 40 μg/ml cycloheximide for 0, 2, 4, 6, 12, or 24 h prior to harvest. Equal volumes of the soluble protein fraction were analyzed by Western blotting to show the increased stability of 16E1^E4 T57D relative to those of the non-T57-phosphorylated WT 16E1^E4 and the GAPDH loading controls. The results shown are typical of results from triplicate experiments. (E) SiHa cells were cotransfected with a plasmid expressing either T57A, T57D, or WT 16E1^E4 and the GFP-expressing plasmid pXJ-GFP (as a transfection control). Cells were harvested at multiple time points posttransfection, as indicated. Total extracts were run on SDS-polyacrylamide gels and analyzed with antibodies against GFP and 16E1^E4 to show the accumulation of 16E1^E4 in the cell. The time course shows the more rapid accumulation of the T57D (phosphomimic) form than of the WT 16E1^E4 (which lacks a T57-phosphorylated form before the 24-h time point) and the 16E1^E4 T57A mutant (which lacks the T57 phosphorylation site). The results shown are typical of results from triplicate experiments. The molecular masses of 16E1^E4 forms (in kilodaltons) are shown to the right (left panel).

    Techniques Used: Incubation, Western Blot, Infection, Staining, Expressing, FACS, Fluorescence, Transfection, Plasmid Preparation, SDS Page, Migration, Mutagenesis

    Effect of 16E5 on ERK and 16E1^E4 phosphorylation. (A) RT-PCR was carried out with a 16E5-expressing SiHa cell line to detect the presence of E5-encoding mRNA. SiHa cells transfected with empty plasmid were used as the negative control. For the positive control, the plasmid pMT3H16E5KC was used as the template for the PCR. (B) E5-positive or E5-negative SiHa cells were infected with rAd16E1^E4 for 24 h and incubated with 10 ng/ml EGF or 0.5 M sorbitol for 10 min prior to harvest. Cell extracts with (+) or without (−) EGF or sorbitol treatment were analyzed by Western blotting with antibodies against active ERK, GAPDH, 16E1^E4, or T57-phosphorylated 16E1^E4 (phospho T57). Molecular mass standards (in kilodaltons) are shown to the left.
    Figure Legend Snippet: Effect of 16E5 on ERK and 16E1^E4 phosphorylation. (A) RT-PCR was carried out with a 16E5-expressing SiHa cell line to detect the presence of E5-encoding mRNA. SiHa cells transfected with empty plasmid were used as the negative control. For the positive control, the plasmid pMT3H16E5KC was used as the template for the PCR. (B) E5-positive or E5-negative SiHa cells were infected with rAd16E1^E4 for 24 h and incubated with 10 ng/ml EGF or 0.5 M sorbitol for 10 min prior to harvest. Cell extracts with (+) or without (−) EGF or sorbitol treatment were analyzed by Western blotting with antibodies against active ERK, GAPDH, 16E1^E4, or T57-phosphorylated 16E1^E4 (phospho T57). Molecular mass standards (in kilodaltons) are shown to the left.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Plasmid Preparation, Negative Control, Positive Control, Polymerase Chain Reaction, Infection, Incubation, Western Blot

    2) Product Images from "Phosphorylation of the Human Papillomavirus Type 16 E1^E4 Protein at T57 by ERK Triggers a Structural Change That Enhances Keratin Binding and Protein Stability ▿Phosphorylation of the Human Papillomavirus Type 16 E1^E4 Protein at T57 by ERK Triggers a Structural Change That Enhances Keratin Binding and Protein Stability ▿ †"

    Article Title: Phosphorylation of the Human Papillomavirus Type 16 E1^E4 Protein at T57 by ERK Triggers a Structural Change That Enhances Keratin Binding and Protein Stability ▿Phosphorylation of the Human Papillomavirus Type 16 E1^E4 Protein at T57 by ERK Triggers a Structural Change That Enhances Keratin Binding and Protein Stability ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.02063-08

    T57 phosphorylation increases the stability and abundance of full-length 16E1^E4 in the cell. (A) His-16E1^E4 was incubated at 30°C in the presence (+) or absence (−) of ERK for 1 h and analyzed by Western blotting using antibodies to 16E1^E4 or to T57-phosphorylated 16E1^E4 (phospho T57). High- and low-level exposures of these blots are shown, demonstrating that the phosphospecific antibody did not detect unphosphorylated E1^E4 (upper panels). SiHa or PHK cells were infected with rAd16E1^E4 for 24 h, and cell lysates were analyzed by Western blotting using antibodies to 16E1^E4 or to T57-phosphorylated 16E1^E4. The antibody to T57-phosphorylated 16E1^E4 detects only the slower-migrating band, confirming that this band is the T57-phosphorylated form of 16E1^E4 (lower panels). (B) SiHa cells were infected with rAd16E1^E4 for 24 or 48 h, fixed with 5% formaldehyde, and triple stained with antibodies to total 16E1^E4 (green) and T57-phosphorylated 16E1^E4 (red) and with DAPI (4′,6-diamidino-2-phenylindole; blue). T57-phosphorylated 16E1^E4 appeared only in cells containing abundant E1^E4 (shown by arrows). Images were captured using a 20× objective. (C) SiHa cells were infected with rAd16E1^E4 for 24 h, and then the SiHa cells expressing high and low levels of E1^E4 were separated by FACS and analyzed by Western blotting with antibodies against 16E1^E4 and GAPDH (as a loading control). Fluorescence intensity correlated with protein abundance and the presence of the T57-phosphorylated (slower-migrating) E1^E4 form. The molecular masses of 16E^E4 forms (in kilodaltons) are shown to the right of the lower panel. +ve, positive; −ve, negative; FITC, fluorescein isothiocyanate. (D, top panel) SiHa cells were transfected with a 16E1^E4 WT-, T57A-, or T57D-expressing plasmid for 24 h prior to SDS-PAGE and Western blotting to reveal the different migration patterns. The molecular masses of 16E1^E4 forms (in kilodaltons) are shown to the left. (Bottom panels) SiHa cells were transfected with a 16E1^E4 WT- or T57D-expressing plasmid for 24 h before being treated with 40 μg/ml cycloheximide for 0, 2, 4, 6, 12, or 24 h prior to harvest. Equal volumes of the soluble protein fraction were analyzed by Western blotting to show the increased stability of 16E1^E4 T57D relative to those of the non-T57-phosphorylated WT 16E1^E4 and the GAPDH loading controls. The results shown are typical of results from triplicate experiments. (E) SiHa cells were cotransfected with a plasmid expressing either T57A, T57D, or WT 16E1^E4 and the GFP-expressing plasmid pXJ-GFP (as a transfection control). Cells were harvested at multiple time points posttransfection, as indicated. Total extracts were run on SDS-polyacrylamide gels and analyzed with antibodies against GFP and 16E1^E4 to show the accumulation of 16E1^E4 in the cell. The time course shows the more rapid accumulation of the T57D (phosphomimic) form than of the WT 16E1^E4 (which lacks a T57-phosphorylated form before the 24-h time point) and the 16E1^E4 T57A mutant (which lacks the T57 phosphorylation site). The results shown are typical of results from triplicate experiments. The molecular masses of 16E1^E4 forms (in kilodaltons) are shown to the right (left panel).
    Figure Legend Snippet: T57 phosphorylation increases the stability and abundance of full-length 16E1^E4 in the cell. (A) His-16E1^E4 was incubated at 30°C in the presence (+) or absence (−) of ERK for 1 h and analyzed by Western blotting using antibodies to 16E1^E4 or to T57-phosphorylated 16E1^E4 (phospho T57). High- and low-level exposures of these blots are shown, demonstrating that the phosphospecific antibody did not detect unphosphorylated E1^E4 (upper panels). SiHa or PHK cells were infected with rAd16E1^E4 for 24 h, and cell lysates were analyzed by Western blotting using antibodies to 16E1^E4 or to T57-phosphorylated 16E1^E4. The antibody to T57-phosphorylated 16E1^E4 detects only the slower-migrating band, confirming that this band is the T57-phosphorylated form of 16E1^E4 (lower panels). (B) SiHa cells were infected with rAd16E1^E4 for 24 or 48 h, fixed with 5% formaldehyde, and triple stained with antibodies to total 16E1^E4 (green) and T57-phosphorylated 16E1^E4 (red) and with DAPI (4′,6-diamidino-2-phenylindole; blue). T57-phosphorylated 16E1^E4 appeared only in cells containing abundant E1^E4 (shown by arrows). Images were captured using a 20× objective. (C) SiHa cells were infected with rAd16E1^E4 for 24 h, and then the SiHa cells expressing high and low levels of E1^E4 were separated by FACS and analyzed by Western blotting with antibodies against 16E1^E4 and GAPDH (as a loading control). Fluorescence intensity correlated with protein abundance and the presence of the T57-phosphorylated (slower-migrating) E1^E4 form. The molecular masses of 16E^E4 forms (in kilodaltons) are shown to the right of the lower panel. +ve, positive; −ve, negative; FITC, fluorescein isothiocyanate. (D, top panel) SiHa cells were transfected with a 16E1^E4 WT-, T57A-, or T57D-expressing plasmid for 24 h prior to SDS-PAGE and Western blotting to reveal the different migration patterns. The molecular masses of 16E1^E4 forms (in kilodaltons) are shown to the left. (Bottom panels) SiHa cells were transfected with a 16E1^E4 WT- or T57D-expressing plasmid for 24 h before being treated with 40 μg/ml cycloheximide for 0, 2, 4, 6, 12, or 24 h prior to harvest. Equal volumes of the soluble protein fraction were analyzed by Western blotting to show the increased stability of 16E1^E4 T57D relative to those of the non-T57-phosphorylated WT 16E1^E4 and the GAPDH loading controls. The results shown are typical of results from triplicate experiments. (E) SiHa cells were cotransfected with a plasmid expressing either T57A, T57D, or WT 16E1^E4 and the GFP-expressing plasmid pXJ-GFP (as a transfection control). Cells were harvested at multiple time points posttransfection, as indicated. Total extracts were run on SDS-polyacrylamide gels and analyzed with antibodies against GFP and 16E1^E4 to show the accumulation of 16E1^E4 in the cell. The time course shows the more rapid accumulation of the T57D (phosphomimic) form than of the WT 16E1^E4 (which lacks a T57-phosphorylated form before the 24-h time point) and the 16E1^E4 T57A mutant (which lacks the T57 phosphorylation site). The results shown are typical of results from triplicate experiments. The molecular masses of 16E1^E4 forms (in kilodaltons) are shown to the right (left panel).

    Techniques Used: Incubation, Western Blot, Infection, Staining, Expressing, FACS, Fluorescence, Transfection, Plasmid Preparation, SDS Page, Migration, Mutagenesis

    16E1^E4 protein is phosphorylated by multiple kinases, with ERK stimulating a gel shift. (A) Putative phosphorylation sites of 16E1^E4 based on common kinase consensus sites are shown. (B) His-16E1^E4 was used as a substrate in kinase assays with [γ- 32 P]ATP and CDK1, CDK2, CKII, ERK, PKA, or PKC α. Samples were separated by SDS-PAGE, and then phosphorylation was detected using a phosphorimager. His-16E1^E4 was phosphorylated by CDK1, CDK2, ERK, PKA, and PKC α. Autophosphorylation (auto-phospho) of the kinase is apparent in the CKII and PKC α lanes. Molecular mass standards (in kilodaltons) are shown to the right. 16E1^E4 phospho, phosphorylated 16E1^E4; +, present; −, absent. (C) His-16E1^E4 was incubated with or without ERK under kinase assay conditions and then analyzed by silver staining. 16E1^E4 phosphorylation by ERK caused a gel shift. Molecular mass standards (in kilodaltons) are shown to the left. (D) rAd16E1^E4-infected SiHa cells were incubated with the kinase inhibitors SB203580, PD98059, and U0126 (separately or in combination) 6 h postinfection. Cells were harvested 24 h postinfection, and extracts were analyzed by Western blotting with anti-16E1^E4 antibody. The MEK inhibitors PD98059 and/or U0126 reduced the intensity of the upper 16E1^E4 band.
    Figure Legend Snippet: 16E1^E4 protein is phosphorylated by multiple kinases, with ERK stimulating a gel shift. (A) Putative phosphorylation sites of 16E1^E4 based on common kinase consensus sites are shown. (B) His-16E1^E4 was used as a substrate in kinase assays with [γ- 32 P]ATP and CDK1, CDK2, CKII, ERK, PKA, or PKC α. Samples were separated by SDS-PAGE, and then phosphorylation was detected using a phosphorimager. His-16E1^E4 was phosphorylated by CDK1, CDK2, ERK, PKA, and PKC α. Autophosphorylation (auto-phospho) of the kinase is apparent in the CKII and PKC α lanes. Molecular mass standards (in kilodaltons) are shown to the right. 16E1^E4 phospho, phosphorylated 16E1^E4; +, present; −, absent. (C) His-16E1^E4 was incubated with or without ERK under kinase assay conditions and then analyzed by silver staining. 16E1^E4 phosphorylation by ERK caused a gel shift. Molecular mass standards (in kilodaltons) are shown to the left. (D) rAd16E1^E4-infected SiHa cells were incubated with the kinase inhibitors SB203580, PD98059, and U0126 (separately or in combination) 6 h postinfection. Cells were harvested 24 h postinfection, and extracts were analyzed by Western blotting with anti-16E1^E4 antibody. The MEK inhibitors PD98059 and/or U0126 reduced the intensity of the upper 16E1^E4 band.

    Techniques Used: Electrophoretic Mobility Shift Assay, SDS Page, Incubation, Kinase Assay, Silver Staining, Infection, Western Blot

    The ERK-mediated gel shift is triggered by the phosphorylation of the threonine residue at consensus site position 57. (A) The His-16E^E4 WT and mutants S43/44A, S49A, T51A, T54A, and T57A were used in nonradioactive kinase assays and detected by SDS-PAGE and silver staining. Only 16E1^E4 T57A (with a mutation in the ERK/MAPK consensus site) failed to show the gel shift. A molecular mass standard (in kilodaltons) is shown to the left. +, present; −, absent. (B) 2D SDS-PAGE and Western blotting show that in the cells transfected with WT or mutant 16E1^E4, WT 16E1^E4 exists as an unphosphorylated protein (pI 9.2) and a singly phosphorylated form (pI 8). A minor multiply phosphorylated species (pI 6.7) increases in abundance in the presence of OA. In the presence of OA (PP2A inhibition), the T57A and S32A mutants, but not mutant S43/44A, failed to produce the second main spot. (C) His-16E1^E4 WT and S32A proteins were analyzed by nonradioactive kinase assays and detected by SDS-PAGE and silver staining. S32A phosphorylation by CDK1 was abolished, and that by CDK2 was clearly reduced. A molecular mass standard (in kilodaltons) is shown to the left.
    Figure Legend Snippet: The ERK-mediated gel shift is triggered by the phosphorylation of the threonine residue at consensus site position 57. (A) The His-16E^E4 WT and mutants S43/44A, S49A, T51A, T54A, and T57A were used in nonradioactive kinase assays and detected by SDS-PAGE and silver staining. Only 16E1^E4 T57A (with a mutation in the ERK/MAPK consensus site) failed to show the gel shift. A molecular mass standard (in kilodaltons) is shown to the left. +, present; −, absent. (B) 2D SDS-PAGE and Western blotting show that in the cells transfected with WT or mutant 16E1^E4, WT 16E1^E4 exists as an unphosphorylated protein (pI 9.2) and a singly phosphorylated form (pI 8). A minor multiply phosphorylated species (pI 6.7) increases in abundance in the presence of OA. In the presence of OA (PP2A inhibition), the T57A and S32A mutants, but not mutant S43/44A, failed to produce the second main spot. (C) His-16E1^E4 WT and S32A proteins were analyzed by nonradioactive kinase assays and detected by SDS-PAGE and silver staining. S32A phosphorylation by CDK1 was abolished, and that by CDK2 was clearly reduced. A molecular mass standard (in kilodaltons) is shown to the left.

    Techniques Used: Electrophoretic Mobility Shift Assay, SDS Page, Silver Staining, Mutagenesis, Western Blot, Transfection, Inhibition

    T57 phosphorylation triggers 16E1^E4 structural change. (A) Dependence of electrophoretic mobilities ( R m ) of the 16E1^E4 phosphorylated (E4-p) and unphosphorylated (E4) species on the acrylamide concentration in SDS-PAGE (Ferguson plot). (B) Intrinsic fluorescence upon the phosphorylation of His-16E1^E4 by ERK was monitored. The fluorescence maxima prior to phosphorylation (time, 0 min; green) and upon the completion of phosphorylation (time, 30 min; red) are indicated on the plot. The decrease in fluorescence intensity and the red shift of the spectrum accompanying phosphorylation are consistent with the tryptophan residues becoming more buried in a hydrophobic environment. (C) The 16E1^E4 protein has elements of secondary structure at its C and N termini (red, α helix; blue, β sheet). T57 is located in a highly charged unstructured region of the protein, and its phosphorylation contributes to the polarization of this region. The effect of T57 phosphorylation on this unstructured region of the protein was assessed using a 27-amino-acid peptide as indicated. (D) The effect of T57 phosphorylation on the structure of the peptide (indicated in panel C) was monitored by recording fluorescent resonance energy transfer (FRET) between the tryptophan residue and a fluorescence acceptor molecule on the terminal cysteine. The excitation (250- to 400-nm) and emission (450- to 600-nm) spectra of the phosphorylated (E4-P) and unphosphorylated (E4) peptides are shown. The peak at 280 nm in the difference spectrum reveals that fluorescence transfer is observed only in the phosphorylated peptide, which indicates that T57 phosphorylation compacts the peptide. (E) Schematic summarizing the effect of T57 phosphorylation by ERK on the structure of 16E1^E4. Upon phosphorylation, the addition of a negative charge on T57 increases the charge polarization in the loop region of the protein and, as indicated by the findings of the tryptophan fluorescence studies, results in additional restraints in this region.
    Figure Legend Snippet: T57 phosphorylation triggers 16E1^E4 structural change. (A) Dependence of electrophoretic mobilities ( R m ) of the 16E1^E4 phosphorylated (E4-p) and unphosphorylated (E4) species on the acrylamide concentration in SDS-PAGE (Ferguson plot). (B) Intrinsic fluorescence upon the phosphorylation of His-16E1^E4 by ERK was monitored. The fluorescence maxima prior to phosphorylation (time, 0 min; green) and upon the completion of phosphorylation (time, 30 min; red) are indicated on the plot. The decrease in fluorescence intensity and the red shift of the spectrum accompanying phosphorylation are consistent with the tryptophan residues becoming more buried in a hydrophobic environment. (C) The 16E1^E4 protein has elements of secondary structure at its C and N termini (red, α helix; blue, β sheet). T57 is located in a highly charged unstructured region of the protein, and its phosphorylation contributes to the polarization of this region. The effect of T57 phosphorylation on this unstructured region of the protein was assessed using a 27-amino-acid peptide as indicated. (D) The effect of T57 phosphorylation on the structure of the peptide (indicated in panel C) was monitored by recording fluorescent resonance energy transfer (FRET) between the tryptophan residue and a fluorescence acceptor molecule on the terminal cysteine. The excitation (250- to 400-nm) and emission (450- to 600-nm) spectra of the phosphorylated (E4-P) and unphosphorylated (E4) peptides are shown. The peak at 280 nm in the difference spectrum reveals that fluorescence transfer is observed only in the phosphorylated peptide, which indicates that T57 phosphorylation compacts the peptide. (E) Schematic summarizing the effect of T57 phosphorylation by ERK on the structure of 16E1^E4. Upon phosphorylation, the addition of a negative charge on T57 increases the charge polarization in the loop region of the protein and, as indicated by the findings of the tryptophan fluorescence studies, results in additional restraints in this region.

    Techniques Used: Concentration Assay, SDS Page, Fluorescence, Förster Resonance Energy Transfer

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    Autoradiography:

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    Blocking Assay:

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    Incubation:

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    Article Snippet: Western Blotting The cells were washed twice with ice-cold phosphate-buffered saline (PBS) and incubated on ice for 30 min with SDS lysis buffer. .. Primary antibodies, including anti-Oct4 (Santa Cruz Biotechnology), anti-Nanog (Abcam), anti-HDAC1 (Sigma), anti-HDAC3 (Cell Signaling), anti-HDAC2 (Santa Cruz Biotechnology), anti-HDAC8 (Santa Cruz Biotechnology), anti-H4/acetyl-H4 (Millipore) anti-H3/acetyl-H3 (Millipore), anti-T/Bry (abcam), anti-Gata4 (Santa Cruz Biotechnology), anti-SMA (Sigma) and anti-GAPDH (Sigma) were used in this study.

    Article Title: Specific Alterations in Astrocyte Properties via the GluA2-GAPDH Complex Associated with Multiple Sclerosis
    Article Snippet: .. This was followed by incubation with primary antibodies of anti-GFAP (1:200, Dako Z0334, Glostrup, Denmark), anti-AQP4 (1:200, Abcam, ab9512, Cambridge, MA, USA), anti-mouse IgG (1:50, Sigma, A9044), anti-Occludin (1:100, Santa Cruz Biotechnology, sc-5562, Dallas, TX, USA), anti-EAAT1 (1:200, Abcam, ab416), anti-EAAT2 (1:200, Abcam, ab41621), anti-GluA2 (1:200, Novus Biologicals, NBP1-46490, Oakville, Canada) and anti-GAPDH (1:200, Millipore Canada, MAB374, Etobicoke, Canada) overnight at 4 °C. .. Alexa 488- or 594-conjugated secondary antibodies (1:200; Thermo Fisher Scientific, Burlington, Canada) in blocking solution were added for 2 hours at room temperature.

    Article Title: GATA4 promotes hepatoblastoma cell proliferation by altering expression of miR125b and DKK3
    Article Snippet: Equal amounts of cell extracts were separated on SDS-PAGE gels, transferred onto nitrocellulose membranes, and incubated with the appropriate antibodies. .. The following antibodies were used: rabbit polyclonal IgG anti-DKK3 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal IgG anti-GATA4 (Santa Cruz Biotechnology), anti-tubulin (Santa Cruz Biotechnology), and anti-GAPDH (Sigma-Aldrich, St. Louis, MO, USA).

    Article Title: The transcription elongation factor TCEA3 induces apoptosis in rhabdomyosarcoma
    Article Snippet: Membranes were again washed with 1X TBST and incubated with chemiluminescent substrate according to the manufacture’s protocol (SuperSignal, Pierce, Rockford, IL) and visualized by autoradiography. .. The other antibodies used included anti-GAPDH (Millipore), anti-BCL2 (D17C4, Cell Signaling), anti-BAX (2772S, Cell Signaling), anti-BAD (9292S, Cell Signaling), anti-p-BAD (Ser136)(4366P, Cell Signaling), anti-AIF (SCBT), anti-APAF1 (SCBT), anti-BID (SCBT), anti-COX 2 (SCBT), anti-caspase 9 (9502, Cell Signaling), anti-cleaved caspase-3 (Asp175) (5A1E, Cell Signaling), anti-caspase 3 (D3RGY, Cell Signaling), anti-caspase 8 (1C12) (9746 S, Cell Signaling), anti-TRAIL (SCBT) and anti-Tubulin (Developmental Studies Hybridoma Bank).

    Article Title: Suppression of miR-708 inhibits the Wnt/β-catenin signaling pathway by activating DKK3 in adult B-all
    Article Snippet: The membranes were blocked with 5% non-fat dry milk and incubated overnight at 4°C with primary antibodies against the following proteins: GSK3β, p-GSK3β (Ser 9), DKK3, β-catenin, cyclin D1, Bcl-2, and Bax-1. .. An anti-GAPDH or β-actin antibody obtained from Sigma-Aldrich was used as a loading control.

    Expressing:

    Article Title: Extracellular ?-Synuclein Leads to Microtubule Destabilization via GSK-3?-Dependent Tau Phosphorylation in PC12 Cells
    Article Snippet: Reagents The following antibodies were used in the current study: anti-phospho Tau (Ser396), anti-phospho GSK-3β (Ser9), anti-GSK-3β, anti-α/β-tubulin (Cell Signalling, Beverly, MA, USA), anti-phospho-GSK-3β (Ty216) (BD Biosciences Pharmingen, NJ, Franklin Lakes, USA), anti-GAPDH, anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA), anti-mouse IgG (GE Health Care UK, Little Chalfont, Buckinghamshire, UK), anti-mouse IgG conjugated with gold particles (Jackson Immunoresearch, West Grove, PA, USA). .. Reagents The following antibodies were used in the current study: anti-phospho Tau (Ser396), anti-phospho GSK-3β (Ser9), anti-GSK-3β, anti-α/β-tubulin (Cell Signalling, Beverly, MA, USA), anti-phospho-GSK-3β (Ty216) (BD Biosciences Pharmingen, NJ, Franklin Lakes, USA), anti-GAPDH, anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA), anti-mouse IgG (GE Health Care UK, Little Chalfont, Buckinghamshire, UK), anti-mouse IgG conjugated with gold particles (Jackson Immunoresearch, West Grove, PA, USA).

    BIA-KA:

    Article Title: Activation of ROCK and MLCK tunes regional stress fiber formation and mechanics via preferential myosin light chain phosphorylation
    Article Snippet: Protein content was measured by bicinchoninic acid (BCA) assay and used to normalize samples to the lowest concentration. .. The following primary antibodies were used: anti-phosphorylated myosin light chain 2 (Thr18/Ser19; Cell Signaling Technology), anti-phosphorylated myosin light chain 2 (Ser19) produced in rabbit or in mouse (both obtained from Cell Signaling Technology), anti-GAPDH (Sigma-Aldrich, St. Louis, MO), mouse anti-MLCK (Sigma-Aldrich, St. Louis, MO) and rabbit anti-MLCK (abcam), anti-ROCK 2 (Sigma-Aldrich, St. Louis, MO), anti-Myc tag (Cell Signaling Technology), and anti-ROCK1 (Cell Signaling Technology).

    Modification:

    Article Title: Extracellular ?-Synuclein Leads to Microtubule Destabilization via GSK-3?-Dependent Tau Phosphorylation in PC12 Cells
    Article Snippet: Reagents The following antibodies were used in the current study: anti-phospho Tau (Ser396), anti-phospho GSK-3β (Ser9), anti-GSK-3β, anti-α/β-tubulin (Cell Signalling, Beverly, MA, USA), anti-phospho-GSK-3β (Ty216) (BD Biosciences Pharmingen, NJ, Franklin Lakes, USA), anti-GAPDH, anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA), anti-mouse IgG (GE Health Care UK, Little Chalfont, Buckinghamshire, UK), anti-mouse IgG conjugated with gold particles (Jackson Immunoresearch, West Grove, PA, USA). .. Cell culture reagents: Dulbecco's Modified Eagle's Medium (DMEM), Fetal Bovine Serum (FBS), Horse Serum (HS), penicillin, streptomycin, G418, L-glutamine, and other reagents such as: deoxyribonuclease I, 3-(4,5-dimethyl-2-tiazolilo)-2,5-diphenyl-2H-tetrazolium bromide (MTT), TRI-reagent, polyethylenoimine (PEI), dimethyl sulfoxide (DMSO), 2′-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1Hbenzimidazoletrihydrochloridehy drate (Hoechst 33258), MES (2-N-(morpholino)ethanesulfonic acid), Colchicine and Paclitaxel (Taxol) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

    Article Title: Profilin-1 deficiency leads to SMAD3 upregulation and impaired 3D outgrowth of breast cancer cells
    Article Snippet: Total cell lysate from 3D culture was prepared by incubating cells at 4 °C in the modified RIPA buffer supplemented with 0.5% SDS and protease/phosphatase inhibitor cocktail (Pierce). .. Sources of different antibodies were: anti-Pfn1 (Abcam), anti-phospho-FAK (Y397) (Invitrogen), anti-Smad3 (Biorad), anti-ERK1/2, anti-phospho-ERK1/2 and anti-phospho-Smad3 (S423/S425) (Cell Signalling), anti-Pfn2 and anti-FAK (Santa Cruz), and anti-GAPDH and anti-Tubulin (Sigma-Aldrich) Immunoblotting concentrations for different antibodies were: 1:3000 for anti-Pfn1, anti-GAPDH and anti-tubulin; 1:500 for anti-Pfn2, anti-ERK1/2, anti-pERK1/2 and anti-FAK; and 1:1000 for anti-Smad3, anti-pSmad3 and anti-pFAK.

    Western Blot:

    Article Title: Histone Deacetylase 1 and 3 Regulate the Mesodermal Lineage Commitment of Mouse Embryonic Stem Cells
    Article Snippet: Paragraph title: Western Blotting ... Primary antibodies, including anti-Oct4 (Santa Cruz Biotechnology), anti-Nanog (Abcam), anti-HDAC1 (Sigma), anti-HDAC3 (Cell Signaling), anti-HDAC2 (Santa Cruz Biotechnology), anti-HDAC8 (Santa Cruz Biotechnology), anti-H4/acetyl-H4 (Millipore) anti-H3/acetyl-H3 (Millipore), anti-T/Bry (abcam), anti-Gata4 (Santa Cruz Biotechnology), anti-SMA (Sigma) and anti-GAPDH (Sigma) were used in this study.

    Article Title: GATA4 promotes hepatoblastoma cell proliferation by altering expression of miR125b and DKK3
    Article Snippet: Paragraph title: Western blot ... The following antibodies were used: rabbit polyclonal IgG anti-DKK3 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal IgG anti-GATA4 (Santa Cruz Biotechnology), anti-tubulin (Santa Cruz Biotechnology), and anti-GAPDH (Sigma-Aldrich, St. Louis, MO, USA).

    Article Title: The transcription elongation factor TCEA3 induces apoptosis in rhabdomyosarcoma
    Article Snippet: Paragraph title: Western blot ... The other antibodies used included anti-GAPDH (Millipore), anti-BCL2 (D17C4, Cell Signaling), anti-BAX (2772S, Cell Signaling), anti-BAD (9292S, Cell Signaling), anti-p-BAD (Ser136)(4366P, Cell Signaling), anti-AIF (SCBT), anti-APAF1 (SCBT), anti-BID (SCBT), anti-COX 2 (SCBT), anti-caspase 9 (9502, Cell Signaling), anti-cleaved caspase-3 (Asp175) (5A1E, Cell Signaling), anti-caspase 3 (D3RGY, Cell Signaling), anti-caspase 8 (1C12) (9746 S, Cell Signaling), anti-TRAIL (SCBT) and anti-Tubulin (Developmental Studies Hybridoma Bank).

    Article Title: Systemic activation of NLRP3 inflammasome and plasma α-synuclein levels are correlated with motor severity and progression in Parkinson’s disease
    Article Snippet: Paragraph title: Western blotting ... Blots were probed with the following primary antibodies: anti-NLRP3 (1:1000, Cell Signaling Technology, Beverly, MA, USA), anti-caspase-1 (1:1000, Adipogen Corporation, CA, USA), anti-IL-1β (1:800, R & D Systems, Minneapolis, USA), and anti-GAPDH (1:5000, Sigma-Aldrich, MO, USA).

    Article Title: Inhibition of autophagy as a new means of improving chemotherapy efficiency in high-LC3B triple-negative breast cancers
    Article Snippet: .. The following antibodies were used for western blotting: anti-AP2A1/adaptin (1:1,000; BD Biosciences, 610502), anti-ACTB/β actin (1:20,000; Sigma-Aldrich, A5441), anti-LC3B (1:1,000; Cell Signaling Technology, 2775), anti-ATG7 (1:1,000; Cell Signaling Technology, 8558), anti-BECN1 (1:1,000; Cell Signaling Technology, 3738), anti-GAPDH (1:1,000; Millipore, MAB374), anti-ATG5 (1:1000; Cosmo Bio, TMD-PH-AT5), anti-YAP1 (1:1,000; Cell Signaling Technology, 4912) and anti-phospho-YAP1 (1:1,000; Cell Signaling Technology, 4911). .. For immunofluorescence studies, the anti-LC3 was from NanoTools (1:50; NanoTools 0231–100/LC3–5F10).

    Article Title: Activation of ROCK and MLCK tunes regional stress fiber formation and mechanics via preferential myosin light chain phosphorylation
    Article Snippet: Paragraph title: Western blots ... The following primary antibodies were used: anti-phosphorylated myosin light chain 2 (Thr18/Ser19; Cell Signaling Technology), anti-phosphorylated myosin light chain 2 (Ser19) produced in rabbit or in mouse (both obtained from Cell Signaling Technology), anti-GAPDH (Sigma-Aldrich, St. Louis, MO), mouse anti-MLCK (Sigma-Aldrich, St. Louis, MO) and rabbit anti-MLCK (abcam), anti-ROCK 2 (Sigma-Aldrich, St. Louis, MO), anti-Myc tag (Cell Signaling Technology), and anti-ROCK1 (Cell Signaling Technology).

    Article Title: Suppression of miR-708 inhibits the Wnt/β-catenin signaling pathway by activating DKK3 in adult B-all
    Article Snippet: Paragraph title: Western blotting analysis ... An anti-GAPDH or β-actin antibody obtained from Sigma-Aldrich was used as a loading control.

    Immunohistochemistry:

    Article Title: Specific Alterations in Astrocyte Properties via the GluA2-GAPDH Complex Associated with Multiple Sclerosis
    Article Snippet: Paragraph title: Immunohistochemistry ... This was followed by incubation with primary antibodies of anti-GFAP (1:200, Dako Z0334, Glostrup, Denmark), anti-AQP4 (1:200, Abcam, ab9512, Cambridge, MA, USA), anti-mouse IgG (1:50, Sigma, A9044), anti-Occludin (1:100, Santa Cruz Biotechnology, sc-5562, Dallas, TX, USA), anti-EAAT1 (1:200, Abcam, ab416), anti-EAAT2 (1:200, Abcam, ab41621), anti-GluA2 (1:200, Novus Biologicals, NBP1-46490, Oakville, Canada) and anti-GAPDH (1:200, Millipore Canada, MAB374, Etobicoke, Canada) overnight at 4 °C.

    Protease Inhibitor:

    Article Title: Nek1 and TAZ Interact to Maintain Normal Levels of Polycystin 2
    Article Snippet: Cells were collected and extracted in lysis buffer (0.5% Triton X-100, 20 mM Tris, pH 7.5, 2 mM magnesium chloride, 1 mM dithiothreitol, 1 mM EGTA, 50 mM β-glycerophosphate, 25 mM sodium fluoride, 1 mM sodium vanadate, 100 μg/ml phenylmethanesulfonylfluoride, and protease inhibitor cocktail [Roche, Indianapolis, IN]). .. Anti-Nek1 (Abnova), anti-Myc (Santa Cruz), anti-GAPDH (Calbiochem), and anti-GST (Santa Cruz) were used for immunoblotting.

    Cell Culture:

    Article Title: Extracellular ?-Synuclein Leads to Microtubule Destabilization via GSK-3?-Dependent Tau Phosphorylation in PC12 Cells
    Article Snippet: Reagents The following antibodies were used in the current study: anti-phospho Tau (Ser396), anti-phospho GSK-3β (Ser9), anti-GSK-3β, anti-α/β-tubulin (Cell Signalling, Beverly, MA, USA), anti-phospho-GSK-3β (Ty216) (BD Biosciences Pharmingen, NJ, Franklin Lakes, USA), anti-GAPDH, anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA), anti-mouse IgG (GE Health Care UK, Little Chalfont, Buckinghamshire, UK), anti-mouse IgG conjugated with gold particles (Jackson Immunoresearch, West Grove, PA, USA). .. Cell culture reagents: Dulbecco's Modified Eagle's Medium (DMEM), Fetal Bovine Serum (FBS), Horse Serum (HS), penicillin, streptomycin, G418, L-glutamine, and other reagents such as: deoxyribonuclease I, 3-(4,5-dimethyl-2-tiazolilo)-2,5-diphenyl-2H-tetrazolium bromide (MTT), TRI-reagent, polyethylenoimine (PEI), dimethyl sulfoxide (DMSO), 2′-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1Hbenzimidazoletrihydrochloridehy drate (Hoechst 33258), MES (2-N-(morpholino)ethanesulfonic acid), Colchicine and Paclitaxel (Taxol) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

    Generated:

    Article Title: Nek1 and TAZ Interact to Maintain Normal Levels of Polycystin 2
    Article Snippet: Anti-Nek1 (Abnova), anti-Myc (Santa Cruz), anti-GAPDH (Calbiochem), and anti-GST (Santa Cruz) were used for immunoblotting. .. Anti-PC2 and anti-TAZ antibodies were generated as described previously.

    other:

    Article Title: A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency
    Article Snippet: Antibodies The following monoclonal antibodies (mabs) were obtained from commercial vendors: anti-actin, anti-EEA1 (Sigma), anti-Na+ K+ -ATPase (Novus-Biologicals), anti-GAPDH (Millipore), anti-human Lamp-1 (BD Transductions), anti-Integrin alpha 11 (R & D system), anti-LC3B (Cell Signaling), anti-transferrin receptor (TfR, clone H68.4; Invitrogen), anti-Lamin and anti-Cathepsin D (Santa Cruz biotechnology).

    Imaging:

    Article Title: GATA4 promotes hepatoblastoma cell proliferation by altering expression of miR125b and DKK3
    Article Snippet: The following antibodies were used: rabbit polyclonal IgG anti-DKK3 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal IgG anti-GATA4 (Santa Cruz Biotechnology), anti-tubulin (Santa Cruz Biotechnology), and anti-GAPDH (Sigma-Aldrich, St. Louis, MO, USA). .. Bands were scanned using a ChemiDoc XRSb Imaging System (Bio-Rad, Hercules, CA, USA).

    Article Title: Systemic activation of NLRP3 inflammasome and plasma α-synuclein levels are correlated with motor severity and progression in Parkinson’s disease
    Article Snippet: Blots were probed with the following primary antibodies: anti-NLRP3 (1:1000, Cell Signaling Technology, Beverly, MA, USA), anti-caspase-1 (1:1000, Adipogen Corporation, CA, USA), anti-IL-1β (1:800, R & D Systems, Minneapolis, USA), and anti-GAPDH (1:5000, Sigma-Aldrich, MO, USA). .. The membranes were scanned and analyzed in a Chemiluminescence Imaging System (ProteinSimple, San Jose, CA, USA).

    Polymerase Chain Reaction:

    Article Title: Extracellular ?-Synuclein Leads to Microtubule Destabilization via GSK-3?-Dependent Tau Phosphorylation in PC12 Cells
    Article Snippet: Reagents The following antibodies were used in the current study: anti-phospho Tau (Ser396), anti-phospho GSK-3β (Ser9), anti-GSK-3β, anti-α/β-tubulin (Cell Signalling, Beverly, MA, USA), anti-phospho-GSK-3β (Ty216) (BD Biosciences Pharmingen, NJ, Franklin Lakes, USA), anti-GAPDH, anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA), anti-mouse IgG (GE Health Care UK, Little Chalfont, Buckinghamshire, UK), anti-mouse IgG conjugated with gold particles (Jackson Immunoresearch, West Grove, PA, USA). .. Reagents The following antibodies were used in the current study: anti-phospho Tau (Ser396), anti-phospho GSK-3β (Ser9), anti-GSK-3β, anti-α/β-tubulin (Cell Signalling, Beverly, MA, USA), anti-phospho-GSK-3β (Ty216) (BD Biosciences Pharmingen, NJ, Franklin Lakes, USA), anti-GAPDH, anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA), anti-mouse IgG (GE Health Care UK, Little Chalfont, Buckinghamshire, UK), anti-mouse IgG conjugated with gold particles (Jackson Immunoresearch, West Grove, PA, USA).

    Binding Assay:

    Article Title: Specific Alterations in Astrocyte Properties via the GluA2-GAPDH Complex Associated with Multiple Sclerosis
    Article Snippet: Free floating sections were initially blocked in 5% fetal bovine serum, 1% Triton X-100, 0.5% Tween 20 and 1% skim milk in 0.1 M PBS for 2 hours at room temperature to reduce non-specific binding. .. This was followed by incubation with primary antibodies of anti-GFAP (1:200, Dako Z0334, Glostrup, Denmark), anti-AQP4 (1:200, Abcam, ab9512, Cambridge, MA, USA), anti-mouse IgG (1:50, Sigma, A9044), anti-Occludin (1:100, Santa Cruz Biotechnology, sc-5562, Dallas, TX, USA), anti-EAAT1 (1:200, Abcam, ab416), anti-EAAT2 (1:200, Abcam, ab41621), anti-GluA2 (1:200, Novus Biologicals, NBP1-46490, Oakville, Canada) and anti-GAPDH (1:200, Millipore Canada, MAB374, Etobicoke, Canada) overnight at 4 °C.

    Immunofluorescence:

    Article Title: Inhibition of autophagy as a new means of improving chemotherapy efficiency in high-LC3B triple-negative breast cancers
    Article Snippet: The following antibodies were used for western blotting: anti-AP2A1/adaptin (1:1,000; BD Biosciences, 610502), anti-ACTB/β actin (1:20,000; Sigma-Aldrich, A5441), anti-LC3B (1:1,000; Cell Signaling Technology, 2775), anti-ATG7 (1:1,000; Cell Signaling Technology, 8558), anti-BECN1 (1:1,000; Cell Signaling Technology, 3738), anti-GAPDH (1:1,000; Millipore, MAB374), anti-ATG5 (1:1000; Cosmo Bio, TMD-PH-AT5), anti-YAP1 (1:1,000; Cell Signaling Technology, 4912) and anti-phospho-YAP1 (1:1,000; Cell Signaling Technology, 4911). .. For immunofluorescence studies, the anti-LC3 was from NanoTools (1:50; NanoTools 0231–100/LC3–5F10).

    MTT Assay:

    Article Title: Extracellular ?-Synuclein Leads to Microtubule Destabilization via GSK-3?-Dependent Tau Phosphorylation in PC12 Cells
    Article Snippet: Reagents The following antibodies were used in the current study: anti-phospho Tau (Ser396), anti-phospho GSK-3β (Ser9), anti-GSK-3β, anti-α/β-tubulin (Cell Signalling, Beverly, MA, USA), anti-phospho-GSK-3β (Ty216) (BD Biosciences Pharmingen, NJ, Franklin Lakes, USA), anti-GAPDH, anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA), anti-mouse IgG (GE Health Care UK, Little Chalfont, Buckinghamshire, UK), anti-mouse IgG conjugated with gold particles (Jackson Immunoresearch, West Grove, PA, USA). .. Cell culture reagents: Dulbecco's Modified Eagle's Medium (DMEM), Fetal Bovine Serum (FBS), Horse Serum (HS), penicillin, streptomycin, G418, L-glutamine, and other reagents such as: deoxyribonuclease I, 3-(4,5-dimethyl-2-tiazolilo)-2,5-diphenyl-2H-tetrazolium bromide (MTT), TRI-reagent, polyethylenoimine (PEI), dimethyl sulfoxide (DMSO), 2′-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1Hbenzimidazoletrihydrochloridehy drate (Hoechst 33258), MES (2-N-(morpholino)ethanesulfonic acid), Colchicine and Paclitaxel (Taxol) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

    Mouse Assay:

    Article Title: In Vivo Correction of COX Deficiency by Activation of the AMPK/PGC-1? Axis
    Article Snippet: Anti-PGC-1α antibody was from Abcam; anti-AMPK and anti-AMPK-P antibodies were from Cell Signaling; anti-COI and -COX5a were from Invitrogen; anti-GAPDH was from Millipore. .. MCK-PGC-1α and ACTA-Cre transgenic mice were obtained from Jackson Laboratory, Bar Harbor, USA.

    Article Title: Specific Alterations in Astrocyte Properties via the GluA2-GAPDH Complex Associated with Multiple Sclerosis
    Article Snippet: Immunohistochemistry Lumbosacral spinal cords from EAE mice of different groups were dissected out, fixed in 4% paraformaldehyde (PFA) overnight at 4 °C, cryoprotected in 30% sucrose and frozen at −80 °C before further processing. .. This was followed by incubation with primary antibodies of anti-GFAP (1:200, Dako Z0334, Glostrup, Denmark), anti-AQP4 (1:200, Abcam, ab9512, Cambridge, MA, USA), anti-mouse IgG (1:50, Sigma, A9044), anti-Occludin (1:100, Santa Cruz Biotechnology, sc-5562, Dallas, TX, USA), anti-EAAT1 (1:200, Abcam, ab416), anti-EAAT2 (1:200, Abcam, ab41621), anti-GluA2 (1:200, Novus Biologicals, NBP1-46490, Oakville, Canada) and anti-GAPDH (1:200, Millipore Canada, MAB374, Etobicoke, Canada) overnight at 4 °C.

    Transgenic Assay:

    Article Title: In Vivo Correction of COX Deficiency by Activation of the AMPK/PGC-1? Axis
    Article Snippet: Anti-PGC-1α antibody was from Abcam; anti-AMPK and anti-AMPK-P antibodies were from Cell Signaling; anti-COI and -COX5a were from Invitrogen; anti-GAPDH was from Millipore. .. MCK-PGC-1α and ACTA-Cre transgenic mice were obtained from Jackson Laboratory, Bar Harbor, USA.

    Protein Extraction:

    Article Title: Profilin-1 deficiency leads to SMAD3 upregulation and impaired 3D outgrowth of breast cancer cells
    Article Snippet: Paragraph title: Protein extraction, immunoblotting ... Sources of different antibodies were: anti-Pfn1 (Abcam), anti-phospho-FAK (Y397) (Invitrogen), anti-Smad3 (Biorad), anti-ERK1/2, anti-phospho-ERK1/2 and anti-phospho-Smad3 (S423/S425) (Cell Signalling), anti-Pfn2 and anti-FAK (Santa Cruz), and anti-GAPDH and anti-Tubulin (Sigma-Aldrich) Immunoblotting concentrations for different antibodies were: 1:3000 for anti-Pfn1, anti-GAPDH and anti-tubulin; 1:500 for anti-Pfn2, anti-ERK1/2, anti-pERK1/2 and anti-FAK; and 1:1000 for anti-Smad3, anti-pSmad3 and anti-pFAK.

    Polyacrylamide Gel Electrophoresis:

    Article Title: The transcription elongation factor TCEA3 induces apoptosis in rhabdomyosarcoma
    Article Snippet: Fifty microgram protein was loaded in each well of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). .. The other antibodies used included anti-GAPDH (Millipore), anti-BCL2 (D17C4, Cell Signaling), anti-BAX (2772S, Cell Signaling), anti-BAD (9292S, Cell Signaling), anti-p-BAD (Ser136)(4366P, Cell Signaling), anti-AIF (SCBT), anti-APAF1 (SCBT), anti-BID (SCBT), anti-COX 2 (SCBT), anti-caspase 9 (9502, Cell Signaling), anti-cleaved caspase-3 (Asp175) (5A1E, Cell Signaling), anti-caspase 3 (D3RGY, Cell Signaling), anti-caspase 8 (1C12) (9746 S, Cell Signaling), anti-TRAIL (SCBT) and anti-Tubulin (Developmental Studies Hybridoma Bank).

    Lysis:

    Article Title: Histone Deacetylase 1 and 3 Regulate the Mesodermal Lineage Commitment of Mouse Embryonic Stem Cells
    Article Snippet: Western Blotting The cells were washed twice with ice-cold phosphate-buffered saline (PBS) and incubated on ice for 30 min with SDS lysis buffer. .. Primary antibodies, including anti-Oct4 (Santa Cruz Biotechnology), anti-Nanog (Abcam), anti-HDAC1 (Sigma), anti-HDAC3 (Cell Signaling), anti-HDAC2 (Santa Cruz Biotechnology), anti-HDAC8 (Santa Cruz Biotechnology), anti-H4/acetyl-H4 (Millipore) anti-H3/acetyl-H3 (Millipore), anti-T/Bry (abcam), anti-Gata4 (Santa Cruz Biotechnology), anti-SMA (Sigma) and anti-GAPDH (Sigma) were used in this study.

    Article Title: GATA4 promotes hepatoblastoma cell proliferation by altering expression of miR125b and DKK3
    Article Snippet: Western blot For western blotting, cells were lysed in lysis buffer containing 50 mM Tris-HCl, 150 mM NaCl, 0.5% NP-40, 1 mM Na3 VO4 , 10 mM NaF, 2 mM PMSF. .. The following antibodies were used: rabbit polyclonal IgG anti-DKK3 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal IgG anti-GATA4 (Santa Cruz Biotechnology), anti-tubulin (Santa Cruz Biotechnology), and anti-GAPDH (Sigma-Aldrich, St. Louis, MO, USA).

    Article Title: Nek1 and TAZ Interact to Maintain Normal Levels of Polycystin 2
    Article Snippet: Cells were collected and extracted in lysis buffer (0.5% Triton X-100, 20 mM Tris, pH 7.5, 2 mM magnesium chloride, 1 mM dithiothreitol, 1 mM EGTA, 50 mM β-glycerophosphate, 25 mM sodium fluoride, 1 mM sodium vanadate, 100 μg/ml phenylmethanesulfonylfluoride, and protease inhibitor cocktail [Roche, Indianapolis, IN]). .. Anti-Nek1 (Abnova), anti-Myc (Santa Cruz), anti-GAPDH (Calbiochem), and anti-GST (Santa Cruz) were used for immunoblotting.

    SDS Page:

    Article Title: Histone Deacetylase 1 and 3 Regulate the Mesodermal Lineage Commitment of Mouse Embryonic Stem Cells
    Article Snippet: Equal amounts of cell lysates were separated by SDS-PAGE. .. Primary antibodies, including anti-Oct4 (Santa Cruz Biotechnology), anti-Nanog (Abcam), anti-HDAC1 (Sigma), anti-HDAC3 (Cell Signaling), anti-HDAC2 (Santa Cruz Biotechnology), anti-HDAC8 (Santa Cruz Biotechnology), anti-H4/acetyl-H4 (Millipore) anti-H3/acetyl-H3 (Millipore), anti-T/Bry (abcam), anti-Gata4 (Santa Cruz Biotechnology), anti-SMA (Sigma) and anti-GAPDH (Sigma) were used in this study.

    Article Title: GATA4 promotes hepatoblastoma cell proliferation by altering expression of miR125b and DKK3
    Article Snippet: Equal amounts of cell extracts were separated on SDS-PAGE gels, transferred onto nitrocellulose membranes, and incubated with the appropriate antibodies. .. The following antibodies were used: rabbit polyclonal IgG anti-DKK3 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal IgG anti-GATA4 (Santa Cruz Biotechnology), anti-tubulin (Santa Cruz Biotechnology), and anti-GAPDH (Sigma-Aldrich, St. Louis, MO, USA).

    Article Title: The transcription elongation factor TCEA3 induces apoptosis in rhabdomyosarcoma
    Article Snippet: Fifty microgram protein was loaded in each well of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). .. The other antibodies used included anti-GAPDH (Millipore), anti-BCL2 (D17C4, Cell Signaling), anti-BAX (2772S, Cell Signaling), anti-BAD (9292S, Cell Signaling), anti-p-BAD (Ser136)(4366P, Cell Signaling), anti-AIF (SCBT), anti-APAF1 (SCBT), anti-BID (SCBT), anti-COX 2 (SCBT), anti-caspase 9 (9502, Cell Signaling), anti-cleaved caspase-3 (Asp175) (5A1E, Cell Signaling), anti-caspase 3 (D3RGY, Cell Signaling), anti-caspase 8 (1C12) (9746 S, Cell Signaling), anti-TRAIL (SCBT) and anti-Tubulin (Developmental Studies Hybridoma Bank).

    Article Title: Suppression of miR-708 inhibits the Wnt/β-catenin signaling pathway by activating DKK3 in adult B-all
    Article Snippet: Western blotting analysis Proteins were extracted from the cells, separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes. .. An anti-GAPDH or β-actin antibody obtained from Sigma-Aldrich was used as a loading control.

    shRNA:

    Article Title: Caveolin-1 Regulates P2Y2 Receptor Signaling during Mechanical Injury in Human 1321N1 Astrocytoma
    Article Snippet: Antibodies and Reagents The following antibodies and reagents were used in this study: anti-phospho-Akt (Ser473) (D9E) (1:2000), anti-Akt (pan) (C67E7) (1:1000), anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204, D13.14.4E) (1:2000), and anti-total p44/42 MAPK (ERK1/2) (1:1000) antibodies from Cell Signaling Technology (Boston, MA, USA); rabbit polyclonal anti-caveolin-1 (1:7500), anti-GAPDH (1:10,000) from Sigma-Aldrich (St. Louis, MO, USA); anti-rabbit IgG-Peroxidase antibody and resazurin sodium salt were obtained from Sigma-Aldrich (St. Louis, MO, USA); suramin hexasodium salt was obtained from Tocris Bioscience (Ellisville, MO, USA). .. Control (SC108080) and human caveolin-1 (SC29241) shRNA lentiviral particles were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Radio Immunoprecipitation:

    Article Title: Activation of ROCK and MLCK tunes regional stress fiber formation and mechanics via preferential myosin light chain phosphorylation
    Article Snippet: Western blots As described previously, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer with phosphatase and protease inhibitors (EMD Millipore, Billerica, MA; ; ). .. The following primary antibodies were used: anti-phosphorylated myosin light chain 2 (Thr18/Ser19; Cell Signaling Technology), anti-phosphorylated myosin light chain 2 (Ser19) produced in rabbit or in mouse (both obtained from Cell Signaling Technology), anti-GAPDH (Sigma-Aldrich, St. Louis, MO), mouse anti-MLCK (Sigma-Aldrich, St. Louis, MO) and rabbit anti-MLCK (abcam), anti-ROCK 2 (Sigma-Aldrich, St. Louis, MO), anti-Myc tag (Cell Signaling Technology), and anti-ROCK1 (Cell Signaling Technology).

    Produced:

    Article Title: Activation of ROCK and MLCK tunes regional stress fiber formation and mechanics via preferential myosin light chain phosphorylation
    Article Snippet: .. The following primary antibodies were used: anti-phosphorylated myosin light chain 2 (Thr18/Ser19; Cell Signaling Technology), anti-phosphorylated myosin light chain 2 (Ser19) produced in rabbit or in mouse (both obtained from Cell Signaling Technology), anti-GAPDH (Sigma-Aldrich, St. Louis, MO), mouse anti-MLCK (Sigma-Aldrich, St. Louis, MO) and rabbit anti-MLCK (abcam), anti-ROCK 2 (Sigma-Aldrich, St. Louis, MO), anti-Myc tag (Cell Signaling Technology), and anti-ROCK1 (Cell Signaling Technology). .. The following secondaries were used: IRDye 800 Goat anti-mouse IgG, IRDye 700 Goat anti-rabbit IgG (Licor), and HRP-conjugated anti-mouse (Life Technologies).

    Concentration Assay:

    Article Title: Activation of ROCK and MLCK tunes regional stress fiber formation and mechanics via preferential myosin light chain phosphorylation
    Article Snippet: Protein content was measured by bicinchoninic acid (BCA) assay and used to normalize samples to the lowest concentration. .. The following primary antibodies were used: anti-phosphorylated myosin light chain 2 (Thr18/Ser19; Cell Signaling Technology), anti-phosphorylated myosin light chain 2 (Ser19) produced in rabbit or in mouse (both obtained from Cell Signaling Technology), anti-GAPDH (Sigma-Aldrich, St. Louis, MO), mouse anti-MLCK (Sigma-Aldrich, St. Louis, MO) and rabbit anti-MLCK (abcam), anti-ROCK 2 (Sigma-Aldrich, St. Louis, MO), anti-Myc tag (Cell Signaling Technology), and anti-ROCK1 (Cell Signaling Technology).

    Staining:

    Article Title: Specific Alterations in Astrocyte Properties via the GluA2-GAPDH Complex Associated with Multiple Sclerosis
    Article Snippet: This was followed by incubation with primary antibodies of anti-GFAP (1:200, Dako Z0334, Glostrup, Denmark), anti-AQP4 (1:200, Abcam, ab9512, Cambridge, MA, USA), anti-mouse IgG (1:50, Sigma, A9044), anti-Occludin (1:100, Santa Cruz Biotechnology, sc-5562, Dallas, TX, USA), anti-EAAT1 (1:200, Abcam, ab416), anti-EAAT2 (1:200, Abcam, ab41621), anti-GluA2 (1:200, Novus Biologicals, NBP1-46490, Oakville, Canada) and anti-GAPDH (1:200, Millipore Canada, MAB374, Etobicoke, Canada) overnight at 4 °C. .. DAPI was used to stain nuclei.

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