Structured Review

Peptide Institute leupeptin
Production of the 90-kDa E-cadherin fragment depends on proteasome and Hakai. (A) Effects of proteasome inhibitors on 90-kDa fragment production. Caco-2 cells were transfected with control or KIFC3-specific siRNA. At 7 h after transfection, cells were passaged. After 24 h, dimethyl sulfoxide, 1 μM MG132, 10 μM lactacystin, or 100 nM epoxomycin was added to culture medium. At 16 h later, cells were harvested and subjected to Western blotting assay using the antibodies indicated. (B) Effects of Hakai depletion on 90-kDa fragment production. At 48 h after transfection of cells with indicated siRNAs, cells were harvested and subjected to Western blotting assay using the antibodies indicated. Knockdown efficiency for each molecule is shown on the far right. (C) Effects of proteasome and lysosome inhibitors on 90-kDa fragment production. Cells were treated with siRNAs and subsequently with 10 mM NH 4 Cl and 21 μM <t>leupeptin</t> with or without 100 nM epoxomycin, according to the same protocol as in A.
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Images

1) Product Images from "Minus end–directed motor KIFC3 suppresses E-cadherin degradation by recruiting USP47 to adherens junctions"

Article Title: Minus end–directed motor KIFC3 suppresses E-cadherin degradation by recruiting USP47 to adherens junctions

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E14-07-1245

Production of the 90-kDa E-cadherin fragment depends on proteasome and Hakai. (A) Effects of proteasome inhibitors on 90-kDa fragment production. Caco-2 cells were transfected with control or KIFC3-specific siRNA. At 7 h after transfection, cells were passaged. After 24 h, dimethyl sulfoxide, 1 μM MG132, 10 μM lactacystin, or 100 nM epoxomycin was added to culture medium. At 16 h later, cells were harvested and subjected to Western blotting assay using the antibodies indicated. (B) Effects of Hakai depletion on 90-kDa fragment production. At 48 h after transfection of cells with indicated siRNAs, cells were harvested and subjected to Western blotting assay using the antibodies indicated. Knockdown efficiency for each molecule is shown on the far right. (C) Effects of proteasome and lysosome inhibitors on 90-kDa fragment production. Cells were treated with siRNAs and subsequently with 10 mM NH 4 Cl and 21 μM leupeptin with or without 100 nM epoxomycin, according to the same protocol as in A.
Figure Legend Snippet: Production of the 90-kDa E-cadherin fragment depends on proteasome and Hakai. (A) Effects of proteasome inhibitors on 90-kDa fragment production. Caco-2 cells were transfected with control or KIFC3-specific siRNA. At 7 h after transfection, cells were passaged. After 24 h, dimethyl sulfoxide, 1 μM MG132, 10 μM lactacystin, or 100 nM epoxomycin was added to culture medium. At 16 h later, cells were harvested and subjected to Western blotting assay using the antibodies indicated. (B) Effects of Hakai depletion on 90-kDa fragment production. At 48 h after transfection of cells with indicated siRNAs, cells were harvested and subjected to Western blotting assay using the antibodies indicated. Knockdown efficiency for each molecule is shown on the far right. (C) Effects of proteasome and lysosome inhibitors on 90-kDa fragment production. Cells were treated with siRNAs and subsequently with 10 mM NH 4 Cl and 21 μM leupeptin with or without 100 nM epoxomycin, according to the same protocol as in A.

Techniques Used: Transfection, Western Blot

2) Product Images from "PTEN Increases Autophagy and Inhibits the Ubiquitin-Proteasome Pathway in Glioma Cells Independently of its Lipid Phosphatase Activity"

Article Title: PTEN Increases Autophagy and Inhibits the Ubiquitin-Proteasome Pathway in Glioma Cells Independently of its Lipid Phosphatase Activity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0083318

Inhibition of the ubiquitin-proteasome pathway and activation of autophagy by PTEN in U87MG cells are independent of mTOR. U87MG cells expressing WT-PTEN or C124S-PTEN ( A ) or WT-PTEN or G129E-PTEN and mock-treated ( B ) were incubated for 18 h with doxycycline and in the last 2 h the following inhibitors were added as indicated: rapamycin (RAP, 200 mM, mTOR inhibitor), KT5720 (25 µM, PKA inhibitor), PD98059 (10 µM, ERK1/2 inhibitor) and SB203580 (10 µM, p38 inhibitor). Before collecting the cells, proteasome (50 µM MG132, A ) and lysosomal (100 µM leupeptin and 20 mM NH 4 Cl, B) inhibitors were also added for 1 h. Total lysates were analyzed by SDS-PAGE and Western blot with antibodies that recognize ubiquitinated proteins (FK1) ( A ), LC3 ( B ) and, as a loading control actin. Molecular weight markers are indicated on the right and in B the position of LC3-I, and LC3-II bands are also shown. The histograms on the right show means ± SD of the densitometric measurements from three different experiments and are expressed as amounts of ubiquitinated proteins (ubiq. prot.) normalized to the levels of actin ( A ) or as LC3II/actin ratios ( B ). Stars indicate statistically significant differences from the values without the corresponding inhibitor treatment at ** p
Figure Legend Snippet: Inhibition of the ubiquitin-proteasome pathway and activation of autophagy by PTEN in U87MG cells are independent of mTOR. U87MG cells expressing WT-PTEN or C124S-PTEN ( A ) or WT-PTEN or G129E-PTEN and mock-treated ( B ) were incubated for 18 h with doxycycline and in the last 2 h the following inhibitors were added as indicated: rapamycin (RAP, 200 mM, mTOR inhibitor), KT5720 (25 µM, PKA inhibitor), PD98059 (10 µM, ERK1/2 inhibitor) and SB203580 (10 µM, p38 inhibitor). Before collecting the cells, proteasome (50 µM MG132, A ) and lysosomal (100 µM leupeptin and 20 mM NH 4 Cl, B) inhibitors were also added for 1 h. Total lysates were analyzed by SDS-PAGE and Western blot with antibodies that recognize ubiquitinated proteins (FK1) ( A ), LC3 ( B ) and, as a loading control actin. Molecular weight markers are indicated on the right and in B the position of LC3-I, and LC3-II bands are also shown. The histograms on the right show means ± SD of the densitometric measurements from three different experiments and are expressed as amounts of ubiquitinated proteins (ubiq. prot.) normalized to the levels of actin ( A ) or as LC3II/actin ratios ( B ). Stars indicate statistically significant differences from the values without the corresponding inhibitor treatment at ** p

Techniques Used: Inhibition, Activation Assay, Expressing, Incubation, SDS Page, Western Blot, Molecular Weight

PTEN expression in U87MG cells increases the formation of autophagosomes. A ) U87MG cells expressing WT-PTEN, C124S-PTEN and G129E-PTEN or mock-treated were incubated in high (H) or low (L) proteolysis media (see Materials and Methods) for 2 h in the presence of lysosomal inhibitors (100 µM leupeptin and 20 mM NH 4 Cl). Extracts (75 µg protein) were analyzed by SDS-PAGE and immunoblot, with low and high exposure (exp.), using an antibody against LC3 and, as a loading control, an antibody that recognizes actin. A representative experiment is shown. The position of LC3-I and LC3-II bands are indicated on the left and molecular weight markers are indicated on the right. The histogram on the right shows the means ± SD of the densitometric analysis of the LC3-II/actin ratios from five different experiments. Stars indicate statistically significant differences from the corresponding (high or low proteolysis conditions) values in mock-treated cells at ** p
Figure Legend Snippet: PTEN expression in U87MG cells increases the formation of autophagosomes. A ) U87MG cells expressing WT-PTEN, C124S-PTEN and G129E-PTEN or mock-treated were incubated in high (H) or low (L) proteolysis media (see Materials and Methods) for 2 h in the presence of lysosomal inhibitors (100 µM leupeptin and 20 mM NH 4 Cl). Extracts (75 µg protein) were analyzed by SDS-PAGE and immunoblot, with low and high exposure (exp.), using an antibody against LC3 and, as a loading control, an antibody that recognizes actin. A representative experiment is shown. The position of LC3-I and LC3-II bands are indicated on the left and molecular weight markers are indicated on the right. The histogram on the right shows the means ± SD of the densitometric analysis of the LC3-II/actin ratios from five different experiments. Stars indicate statistically significant differences from the corresponding (high or low proteolysis conditions) values in mock-treated cells at ** p

Techniques Used: Expressing, Incubation, SDS Page, Molecular Weight

PTEN expression in U87MG cells increases the formation of autophagosomes under various proteolysis conditions. U87MG cells expressing WT-PTEN, G129E-PTEN or C124S-PTEN ( A ) and mock-treated U87MG cells or expressing WT-PTEN or C124S-PTEN ( B ) were incubated under high (KH), intermediate (Ins or EAA)( A and B ) and low (Ins/EAA)(only in A ) proteolysis conditions for 2 h as in Figure 5C in the presence of lysosomal inhibitors (100 µM leupeptin and 20 mM NH 4 Cl). Extracts (75 µg protein) were analyzed by SDS-PAGE and immunoblot with antibodies that recognize LC3 and, as a loading control, actin. The position of LC3-I and LC3-II bands are indicated on the left and molecular weight markers are indicated on the right. The Western blots on the left show representative experiments and the histograms on the right show the means ± SD of the densitometric analysis of the LC3-II/ratios from five different experiments. Stars indicate statistically significant differences from the corresponding (high, intermediate or low proteolysis conditions) values in the cells expressing the C124S PTEN mutant ( A ) and mock-treated cells ( B ) at ** p
Figure Legend Snippet: PTEN expression in U87MG cells increases the formation of autophagosomes under various proteolysis conditions. U87MG cells expressing WT-PTEN, G129E-PTEN or C124S-PTEN ( A ) and mock-treated U87MG cells or expressing WT-PTEN or C124S-PTEN ( B ) were incubated under high (KH), intermediate (Ins or EAA)( A and B ) and low (Ins/EAA)(only in A ) proteolysis conditions for 2 h as in Figure 5C in the presence of lysosomal inhibitors (100 µM leupeptin and 20 mM NH 4 Cl). Extracts (75 µg protein) were analyzed by SDS-PAGE and immunoblot with antibodies that recognize LC3 and, as a loading control, actin. The position of LC3-I and LC3-II bands are indicated on the left and molecular weight markers are indicated on the right. The Western blots on the left show representative experiments and the histograms on the right show the means ± SD of the densitometric analysis of the LC3-II/ratios from five different experiments. Stars indicate statistically significant differences from the corresponding (high, intermediate or low proteolysis conditions) values in the cells expressing the C124S PTEN mutant ( A ) and mock-treated cells ( B ) at ** p

Techniques Used: Expressing, Incubation, SDS Page, Molecular Weight, Western Blot, Mutagenesis

3) Product Images from "The Regulatory Domain of Human Tryptophan Hydroxylase 1 Forms a Stable Dimer"

Article Title: The Regulatory Domain of Human Tryptophan Hydroxylase 1 Forms a Stable Dimer

Journal: Biochemical and biophysical research communications

doi: 10.1016/j.bbrc.2016.05.144

Gel filtration of RDTPH1 C64S. Conditions: 100 mM NaCl, 1 µM pepstatin A, 1 µM leupeptin, 50 mM phosphate, pH 8.0, 23 °C. Inset: elution volumes of the standard proteins aprotinin (6,500 Da), ribonuclease A (13,700 Da), carbonic
Figure Legend Snippet: Gel filtration of RDTPH1 C64S. Conditions: 100 mM NaCl, 1 µM pepstatin A, 1 µM leupeptin, 50 mM phosphate, pH 8.0, 23 °C. Inset: elution volumes of the standard proteins aprotinin (6,500 Da), ribonuclease A (13,700 Da), carbonic

Techniques Used: Filtration

4) Product Images from "Phosphorylation of Fibroblast Growth Factor (FGF) Receptor 1 at Ser777 by p38 Mitogen-Activated Protein Kinase Regulates Translocation of Exogenous FGF1 to the Cytosol and Nucleus "

Article Title: Phosphorylation of Fibroblast Growth Factor (FGF) Receptor 1 at Ser777 by p38 Mitogen-Activated Protein Kinase Regulates Translocation of Exogenous FGF1 to the Cytosol and Nucleus

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.02117-07

Effect of activation and inhibition of p38 MAPK on endocytosis and intracellular sorting of FGF1. (A) NIH 3T3 cells were incubated with SB203580, SB202474, anisomycin, or PD169316 and increasing concentrations of 125 I-labeled FGF1 for 3 h at 4°C. The cells were washed with 1 M NaCl and lysed, and the solubilized radioactivity, representing the cell surface FGFR-bound 125 I-FGF1, was measured (reported as counts per minute [CPM]). (B) HeLa cells transfected with FGFR1 or FGFR4 were incubated with Cy3-FGF1 and heparin in the presence or absence of SB203580 (10 μM) or anisomycin (10 μM) for 2 h at 37°C in DMEM with 0.3 mM leupeptin. The cells were fixed, stained for LAMP-1 (Cy2), and examined by confocal microscopy. Bar, 5 μm. Images shown are merged images (red plus green) where a section of each image has been magnified and shown to the left as red, green, and merged images separately. The proportion of Cy3-positive structures that colocalized with Cy2-positive structures (indicated by yellow in the merged images) was calculated and presented as the mean from 15 cells in each case. Error bars indicate the standard deviations. (C) NIH 3T3 cells were preincubated with or without 20 μM anisomycin for 15 min at 37°C and then incubated with 35 S-FGF1 and heparin at 4°C for 2 h. The cells were washed with 1 M NaCl and lysed to measure surface-bound FGF1 or, to measure endocytosis, incubated further for 0, 15, 30, or 60 min at 37°C in the presence and absence of 20 μM anisomycin. 35 S-FGF1 was extracted from cell lysates and subjected to SDS-PAGE and fluorography. *, partially degraded FGF1.
Figure Legend Snippet: Effect of activation and inhibition of p38 MAPK on endocytosis and intracellular sorting of FGF1. (A) NIH 3T3 cells were incubated with SB203580, SB202474, anisomycin, or PD169316 and increasing concentrations of 125 I-labeled FGF1 for 3 h at 4°C. The cells were washed with 1 M NaCl and lysed, and the solubilized radioactivity, representing the cell surface FGFR-bound 125 I-FGF1, was measured (reported as counts per minute [CPM]). (B) HeLa cells transfected with FGFR1 or FGFR4 were incubated with Cy3-FGF1 and heparin in the presence or absence of SB203580 (10 μM) or anisomycin (10 μM) for 2 h at 37°C in DMEM with 0.3 mM leupeptin. The cells were fixed, stained for LAMP-1 (Cy2), and examined by confocal microscopy. Bar, 5 μm. Images shown are merged images (red plus green) where a section of each image has been magnified and shown to the left as red, green, and merged images separately. The proportion of Cy3-positive structures that colocalized with Cy2-positive structures (indicated by yellow in the merged images) was calculated and presented as the mean from 15 cells in each case. Error bars indicate the standard deviations. (C) NIH 3T3 cells were preincubated with or without 20 μM anisomycin for 15 min at 37°C and then incubated with 35 S-FGF1 and heparin at 4°C for 2 h. The cells were washed with 1 M NaCl and lysed to measure surface-bound FGF1 or, to measure endocytosis, incubated further for 0, 15, 30, or 60 min at 37°C in the presence and absence of 20 μM anisomycin. 35 S-FGF1 was extracted from cell lysates and subjected to SDS-PAGE and fluorography. *, partially degraded FGF1.

Techniques Used: Activation Assay, Inhibition, Incubation, Labeling, Radioactivity, Transfection, Staining, Confocal Microscopy, SDS Page

5) Product Images from "Transgenic expression of a ratiometric autophagy probe specifically in neurons enables the interrogation of brain autophagy in vivo"

Article Title: Transgenic expression of a ratiometric autophagy probe specifically in neurons enables the interrogation of brain autophagy in vivo

Journal: Autophagy

doi: 10.1080/15548627.2018.1528812

tfLC3 color change in vivo in the brain under lysosomal proteolysis inhibition. Representative fluorescence images of tfLC3 color change in neurons of TRGL6 mouse brain. ( A ) Inhibition of lysosomal proteolysis by leupeptin (Leup) increases the number of yellow puncta in perikarya. Quantitative data analysis shows that yellow signals are increased, and red signals are decreased in Leup-infused (7 d) TRGL6 mouse neurons. Scale bar: 20 μm. Bar colors denote the colors of puncta. n = 28, *** P
Figure Legend Snippet: tfLC3 color change in vivo in the brain under lysosomal proteolysis inhibition. Representative fluorescence images of tfLC3 color change in neurons of TRGL6 mouse brain. ( A ) Inhibition of lysosomal proteolysis by leupeptin (Leup) increases the number of yellow puncta in perikarya. Quantitative data analysis shows that yellow signals are increased, and red signals are decreased in Leup-infused (7 d) TRGL6 mouse neurons. Scale bar: 20 μm. Bar colors denote the colors of puncta. n = 28, *** P

Techniques Used: In Vivo, Inhibition, Fluorescence

6) Product Images from "The Amino Acid Specificity for Activation of Phenylalanine Hydroxylase Matches the Specificity for Stabilization of Regulatory Domain Dimers"

Article Title: The Amino Acid Specificity for Activation of Phenylalanine Hydroxylase Matches the Specificity for Stabilization of Regulatory Domain Dimers

Journal: Biochemistry

doi: 10.1021/acs.biochem.5b00616

Effects of selected amino acids on the 2D 1 H– 15 N HSQC spectra of RDPheH 25–117 : 400 μM RDPheH 25–117 (black) and 400 μM RDPheH 25–117 (red) with (A) 10 mM d -phenylalanine, (B) 10 mM methionine, (C) 10 mM l -4-aminophenylalanine, and (D) 50 mM alanine. The two regions of the spectra shown are the same as those in Figure 2 C. Conditions: 50 mM sodium phosphate, 100 mM NaCl, 1 μM leupeptin, 1 μM pepstatin A, and 10% D 2 O (pH 8.0), at 300 K at a magnetic field strength of 11.7 T (500 MHz for 1 H).
Figure Legend Snippet: Effects of selected amino acids on the 2D 1 H– 15 N HSQC spectra of RDPheH 25–117 : 400 μM RDPheH 25–117 (black) and 400 μM RDPheH 25–117 (red) with (A) 10 mM d -phenylalanine, (B) 10 mM methionine, (C) 10 mM l -4-aminophenylalanine, and (D) 50 mM alanine. The two regions of the spectra shown are the same as those in Figure 2 C. Conditions: 50 mM sodium phosphate, 100 mM NaCl, 1 μM leupeptin, 1 μM pepstatin A, and 10% D 2 O (pH 8.0), at 300 K at a magnetic field strength of 11.7 T (500 MHz for 1 H).

Techniques Used:

Effect of N-terminal deletion on the 1 H– 15 N HSQC NMR spectrum of the regulatory domain of PheH: (A) 1 mM RDPheH and (B) 430 μM RDPheH 25–117 . Conditions: 50 mM sodium phosphate, 100 mM NaCl, 1 μM leupeptin, 1 μM pepstatin A, and 5% D 2 O (pH 8.0), at 300 K at a magnetic field strength of 14.1 T (600 MHz for 1 H).
Figure Legend Snippet: Effect of N-terminal deletion on the 1 H– 15 N HSQC NMR spectrum of the regulatory domain of PheH: (A) 1 mM RDPheH and (B) 430 μM RDPheH 25–117 . Conditions: 50 mM sodium phosphate, 100 mM NaCl, 1 μM leupeptin, 1 μM pepstatin A, and 5% D 2 O (pH 8.0), at 300 K at a magnetic field strength of 14.1 T (600 MHz for 1 H).

Techniques Used: Nuclear Magnetic Resonance

7) Product Images from "Reduction of Brain ?-Amyloid (A?) by Fluvastatin, a Hydroxymethylglutaryl-CoA Reductase Inhibitor, through Increase in Degradation of Amyloid Precursor Protein C-terminal Fragments (APP-CTFs) and A? Clearance *"

Article Title: Reduction of Brain ?-Amyloid (A?) by Fluvastatin, a Hydroxymethylglutaryl-CoA Reductase Inhibitor, through Increase in Degradation of Amyloid Precursor Protein C-terminal Fragments (APP-CTFs) and A? Clearance *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.102277

Effects of lysosomal inhibitors on reduction of APP-CTF and Aβ levels by fluvastatin in brain. A , after mice were treated with or without 5 mg/kg/day fluvastatin for 4 weeks, leupeptin or E64 was administered for 7 days into the cerebral ventricle
Figure Legend Snippet: Effects of lysosomal inhibitors on reduction of APP-CTF and Aβ levels by fluvastatin in brain. A , after mice were treated with or without 5 mg/kg/day fluvastatin for 4 weeks, leupeptin or E64 was administered for 7 days into the cerebral ventricle

Techniques Used: Mouse Assay

8) Product Images from "Autophagic activity measured in whole rat hepatocytes as the accumulation of a novel BHMT fragment (p10), generated in amphisomes by the asparaginyl proteinase, legumain"

Article Title: Autophagic activity measured in whole rat hepatocytes as the accumulation of a novel BHMT fragment (p10), generated in amphisomes by the asparaginyl proteinase, legumain

Journal: Autophagy

doi: 10.4161/auto.7.9.16436

Effect of AJN-230 on autophagic BHMT processing and LDH accumulation in live cells. Rat hepatocytes were incubated at 37°C for up to 4 h in the presence of leupeptin (0.3 mM) and/or AJN-230 at the concentration indicated. The cells were then electrodisrupted and the cell corpses were isolated and solubilized to measure their contents of sedimentable BHMT (by immunoblotting) or lactic dehydrogenase (LDH; as enzymatic activity or by immunoblotting). (A) Inhibition of leupeptin-induced p10 and p32 BHMT formation by the legumain inhibitor, AJN-230. Note the accumulation of a p42 intermediate (upper parts) in parallel with the disappearance of p10 (second part row) and p32 (third part row). In the bottom part row, immunoblotting was done with an internal BHMT antibody that gives a lower background of sedimentable p45. Parts in the same row are from the same gel. (B) Accumulation of large BHMT forms (immunoblotted with the internal BHMT antibody) and LDH in cell corpses during a 4 h incubation of hepatocytes at 37°C in the absence or presence of leupeptin and AJN-230 as indicated. (C) Quantification of LDH accumulation in corpses from cells incubated for 4 h at 37°C without additions (open circles), with leupeptin (LPT) only (0.3 mM, filled circles, whole line) or with leupeptin + AJN-230 (50 µM; filled triangles, dotted line), measured as enzymatic activity in part a or as immunoblotted protein in (b). BHMT accumulation (part b) was measured as the sum of immunoblotted p42 and p45 in cell corpses incubated with leupeptin only (open squares, whole line) or with leupeptin + AJN-230 (filled squares, broken line). Values in part a are the means ± range of two experiments; values in (b) are the means ± SE/range of two to four experiments.
Figure Legend Snippet: Effect of AJN-230 on autophagic BHMT processing and LDH accumulation in live cells. Rat hepatocytes were incubated at 37°C for up to 4 h in the presence of leupeptin (0.3 mM) and/or AJN-230 at the concentration indicated. The cells were then electrodisrupted and the cell corpses were isolated and solubilized to measure their contents of sedimentable BHMT (by immunoblotting) or lactic dehydrogenase (LDH; as enzymatic activity or by immunoblotting). (A) Inhibition of leupeptin-induced p10 and p32 BHMT formation by the legumain inhibitor, AJN-230. Note the accumulation of a p42 intermediate (upper parts) in parallel with the disappearance of p10 (second part row) and p32 (third part row). In the bottom part row, immunoblotting was done with an internal BHMT antibody that gives a lower background of sedimentable p45. Parts in the same row are from the same gel. (B) Accumulation of large BHMT forms (immunoblotted with the internal BHMT antibody) and LDH in cell corpses during a 4 h incubation of hepatocytes at 37°C in the absence or presence of leupeptin and AJN-230 as indicated. (C) Quantification of LDH accumulation in corpses from cells incubated for 4 h at 37°C without additions (open circles), with leupeptin (LPT) only (0.3 mM, filled circles, whole line) or with leupeptin + AJN-230 (50 µM; filled triangles, dotted line), measured as enzymatic activity in part a or as immunoblotted protein in (b). BHMT accumulation (part b) was measured as the sum of immunoblotted p42 and p45 in cell corpses incubated with leupeptin only (open squares, whole line) or with leupeptin + AJN-230 (filled squares, broken line). Values in part a are the means ± range of two experiments; values in (b) are the means ± SE/range of two to four experiments.

Techniques Used: Incubation, Concentration Assay, Isolation, Activity Assay, Inhibition

Secondary formation of BHMT fragments by in vitro proteolysis. (A) Hepatocytes were solubilized directly (as whole cells) in an SDS-containing lysis buffer with or without leupeptin (0.3 mM) as indicated. (B–D) Frozen/thawed hepatocyte homogenates were incubated at 0°C for up to 2 h (B) without additions; (C) with a commercial proteinase inhibitor mixture or (D) with the individual components of the mixture. In a separate experiment (last two lanes), the concentration of AEBSF was trebled (to 3 mM) relative to the mixture and the legumain inhibitor, AJN-230, was tested. All samples were finally solubilized in an SDS-containing lysis buffer for 1 h at 0°C in the presence of leupeptin (0.3 mM), subjected to SDS-PAGE and immunoblotted with the N-terminal BHMT antibody. (E) Effect of legumain and legumain inhibitors on in vitro BHMT cleavage. Frozen/thawed hepatocyte homogenates were acidified to pH 5.6 (by the addition of HCl) and incubated for 16 h at 0°C with the addition of partially purified bovine kidney legumain (100 ng/ml), AEBSF (3 mM), MV-026630 (45 µM) or AJN-230 (75 µM) as indicated. The homogenates were then solubilized in SDS and electrophoresed as described above and immunoblotted with the N-terminal BHMT antibody (upper part) as well as with a C-terminal antibody (lower part) to detect both the N-terminal p10 fragment and its C-terminal cleavage partner, p32.
Figure Legend Snippet: Secondary formation of BHMT fragments by in vitro proteolysis. (A) Hepatocytes were solubilized directly (as whole cells) in an SDS-containing lysis buffer with or without leupeptin (0.3 mM) as indicated. (B–D) Frozen/thawed hepatocyte homogenates were incubated at 0°C for up to 2 h (B) without additions; (C) with a commercial proteinase inhibitor mixture or (D) with the individual components of the mixture. In a separate experiment (last two lanes), the concentration of AEBSF was trebled (to 3 mM) relative to the mixture and the legumain inhibitor, AJN-230, was tested. All samples were finally solubilized in an SDS-containing lysis buffer for 1 h at 0°C in the presence of leupeptin (0.3 mM), subjected to SDS-PAGE and immunoblotted with the N-terminal BHMT antibody. (E) Effect of legumain and legumain inhibitors on in vitro BHMT cleavage. Frozen/thawed hepatocyte homogenates were acidified to pH 5.6 (by the addition of HCl) and incubated for 16 h at 0°C with the addition of partially purified bovine kidney legumain (100 ng/ml), AEBSF (3 mM), MV-026630 (45 µM) or AJN-230 (75 µM) as indicated. The homogenates were then solubilized in SDS and electrophoresed as described above and immunoblotted with the N-terminal BHMT antibody (upper part) as well as with a C-terminal antibody (lower part) to detect both the N-terminal p10 fragment and its C-terminal cleavage partner, p32.

Techniques Used: In Vitro, Lysis, Incubation, Concentration Assay, SDS Page, Purification

Time-dependent accumulation of the p10 BHMT fragment in leupeptin-treated live hepatocytes. (A) Isolated rat hepatocytes were incubated at 37°C for up to 4 h in a suspension buffer containing an energy substrate (pyruvate) only (control) or with the addition of 3-methyladenine (3MA, 10 mM), leupeptin (0.3 mM) or vinblastine (50 µM) alone or in combinations as indicated. The cells were then electrodisrupted and cytosol-free, sedimentable cell corpses prepared. Disruptates (representing whole cells) and cell corpses (containing organelles and the cytoskeleton) were solubilized and immunoblotted with the N-terminal BHMT antibody. Only the p10 fragment is shown. In a separate experiment (bottom parts), immmunoblotting with a C-terminal BHMT antibody was used to demonstrate the leupeptin-induced accumulation of p10s C-terminal sister fragment, p32. (B) Hepatocytes were incubated for up to 2 h at 37°C with leupeptin (Lpt, 0.3 mM) and/or propylamine (Prop, 10 mM) as indicated and cell corpse extracts were immunoblotted with the N-terminal BHMT antibody. Only the p10 fragment is shown. (C) Hepatocytes were incubated for up to 4 h at 37°C with leupeptin (0.3 mM) and various concentrations of AJN-230 as indicated; in the last lane with leupeptin, AJN-230 (50 µM) and 3MA (10 mM). Cell corpse extracts were immunoblotted with the N-terminal BHMT antibody. The whole gel is shown to illustrate the weak bands at 27, 30 and 33 kDa; the last of these will probably not be visible in print.
Figure Legend Snippet: Time-dependent accumulation of the p10 BHMT fragment in leupeptin-treated live hepatocytes. (A) Isolated rat hepatocytes were incubated at 37°C for up to 4 h in a suspension buffer containing an energy substrate (pyruvate) only (control) or with the addition of 3-methyladenine (3MA, 10 mM), leupeptin (0.3 mM) or vinblastine (50 µM) alone or in combinations as indicated. The cells were then electrodisrupted and cytosol-free, sedimentable cell corpses prepared. Disruptates (representing whole cells) and cell corpses (containing organelles and the cytoskeleton) were solubilized and immunoblotted with the N-terminal BHMT antibody. Only the p10 fragment is shown. In a separate experiment (bottom parts), immmunoblotting with a C-terminal BHMT antibody was used to demonstrate the leupeptin-induced accumulation of p10s C-terminal sister fragment, p32. (B) Hepatocytes were incubated for up to 2 h at 37°C with leupeptin (Lpt, 0.3 mM) and/or propylamine (Prop, 10 mM) as indicated and cell corpse extracts were immunoblotted with the N-terminal BHMT antibody. Only the p10 fragment is shown. (C) Hepatocytes were incubated for up to 4 h at 37°C with leupeptin (0.3 mM) and various concentrations of AJN-230 as indicated; in the last lane with leupeptin, AJN-230 (50 µM) and 3MA (10 mM). Cell corpse extracts were immunoblotted with the N-terminal BHMT antibody. The whole gel is shown to illustrate the weak bands at 27, 30 and 33 kDa; the last of these will probably not be visible in print.

Techniques Used: Isolation, Incubation

Effects of AJN-230 and autophagic-lysosomal pathway inhibitors on autophagic sequestration and processing of BHMT and LDH in live cells. Hepatocytes were incubated at 37°C for up to 4 h (A and B) or for 2 × 90 min (C) in the presence of asparagine (ASN; 20 mM), leupeptin (LPT; 0.3 mM), 3-methyladenine (3MA; 10 mM), AJN-230 (AJN; 50 µM) or ammonium chloride (NH 3 ; 20 mM) in various combinations. Cell corpse-associated LDH (top rows), BHMT forms (N-terminal antibody; second and third rows), legumain (fourth row) or cathepsin B (bottom row) were measured by immunoblotting. (A) Effect of asparagine on LDH accumulation and p10 fragment generation. Parts in the same row are from the same gel. (B) Effects of AJN-230 and asparagine on LDH accumulation and p42 accumulation. Parts b,f and j represent a different experiment than the other parts. (C) Inhibition of legumain activity by ammonia. Hepatocytes were first incubated for 90 min at 37°C with ammonium chloride, asparagine or leupeptin as indicated, then washed and reincubated for another 90 min at 37°C with ammonium chloride, leupeptin or 3MA as indicated. Samples from two different experiments (0–90 and 90–180, respectively) were applied to the same gel. (D) Schematic summary of autophagic BHMT and LDH processing. The results of the present study indicate that BHMT is processed in amphisomes: An initial modification (e.g., a C-terminal truncation of full-length p45 BHMT by an unknown, leupeptin-resistant proteinase produces a 42 kDa intermediate that is cleaved by legumain to generate an N-terminal 10-kDa and a C-terminal 32-kDa fragment, both being degraded by leupeptin-sensitive cysteine cathepsins. In addition, p45 and p42 can (sequentially or independently) be degraded by a legumain-independent, leupeptin-sensitive pathway. LDH is degraded in amphisomes by leupeptin-sensitive proteolysis and in lysosomes by leupeptin-resistant proteolysis. Both leupeptin and asparagine suppress amphisome-lysosome fusion, whereas vinblastine (VBL) inhibits the fusion of autophagosomes with later autophagic vacuoles. Ammonia (NH3) reversibly inhibits acid proteolysis in general by neutralizing amphisomes and lysosomes; in addition, the neutralization apparently causes irreversible inhibition of legumain.
Figure Legend Snippet: Effects of AJN-230 and autophagic-lysosomal pathway inhibitors on autophagic sequestration and processing of BHMT and LDH in live cells. Hepatocytes were incubated at 37°C for up to 4 h (A and B) or for 2 × 90 min (C) in the presence of asparagine (ASN; 20 mM), leupeptin (LPT; 0.3 mM), 3-methyladenine (3MA; 10 mM), AJN-230 (AJN; 50 µM) or ammonium chloride (NH 3 ; 20 mM) in various combinations. Cell corpse-associated LDH (top rows), BHMT forms (N-terminal antibody; second and third rows), legumain (fourth row) or cathepsin B (bottom row) were measured by immunoblotting. (A) Effect of asparagine on LDH accumulation and p10 fragment generation. Parts in the same row are from the same gel. (B) Effects of AJN-230 and asparagine on LDH accumulation and p42 accumulation. Parts b,f and j represent a different experiment than the other parts. (C) Inhibition of legumain activity by ammonia. Hepatocytes were first incubated for 90 min at 37°C with ammonium chloride, asparagine or leupeptin as indicated, then washed and reincubated for another 90 min at 37°C with ammonium chloride, leupeptin or 3MA as indicated. Samples from two different experiments (0–90 and 90–180, respectively) were applied to the same gel. (D) Schematic summary of autophagic BHMT and LDH processing. The results of the present study indicate that BHMT is processed in amphisomes: An initial modification (e.g., a C-terminal truncation of full-length p45 BHMT by an unknown, leupeptin-resistant proteinase produces a 42 kDa intermediate that is cleaved by legumain to generate an N-terminal 10-kDa and a C-terminal 32-kDa fragment, both being degraded by leupeptin-sensitive cysteine cathepsins. In addition, p45 and p42 can (sequentially or independently) be degraded by a legumain-independent, leupeptin-sensitive pathway. LDH is degraded in amphisomes by leupeptin-sensitive proteolysis and in lysosomes by leupeptin-resistant proteolysis. Both leupeptin and asparagine suppress amphisome-lysosome fusion, whereas vinblastine (VBL) inhibits the fusion of autophagosomes with later autophagic vacuoles. Ammonia (NH3) reversibly inhibits acid proteolysis in general by neutralizing amphisomes and lysosomes; in addition, the neutralization apparently causes irreversible inhibition of legumain.

Techniques Used: Incubation, Inhibition, Activity Assay, Modification, Neutralization

9) Product Images from "Transgenic expression of a ratiometric autophagy probe specifically in neurons enables the interrogation of brain autophagy in vivo"

Article Title: Transgenic expression of a ratiometric autophagy probe specifically in neurons enables the interrogation of brain autophagy in vivo

Journal: Autophagy

doi: 10.1080/15548627.2018.1528812

tfLC3 color change in vivo in the brain under lysosomal proteolysis inhibition. Representative fluorescence images of tfLC3 color change in neurons of TRGL6 mouse brain. ( A ) Inhibition of lysosomal proteolysis by leupeptin (Leup) increases the number of yellow puncta in perikarya. Quantitative data analysis shows that yellow signals are increased, and red signals are decreased in Leup-infused (7 d) TRGL6 mouse neurons. Scale bar: 20 μm. Bar colors denote the colors of puncta. n = 28, *** PÂÂ
Figure Legend Snippet: tfLC3 color change in vivo in the brain under lysosomal proteolysis inhibition. Representative fluorescence images of tfLC3 color change in neurons of TRGL6 mouse brain. ( A ) Inhibition of lysosomal proteolysis by leupeptin (Leup) increases the number of yellow puncta in perikarya. Quantitative data analysis shows that yellow signals are increased, and red signals are decreased in Leup-infused (7 d) TRGL6 mouse neurons. Scale bar: 20 μm. Bar colors denote the colors of puncta. n = 28, *** PÂÂ

Techniques Used: In Vivo, Inhibition, Fluorescence

10) Product Images from "Degradation of altered mitochondria by autophagy is impaired in Lafora disease"

Article Title: Degradation of altered mitochondria by autophagy is impaired in Lafora disease

Journal: The FEBS journal

doi: 10.1111/febs.14468

Number of autophagic vacuoles in control and in LD fibroblasts in the presence or absence of CCCP Fibroblasts from controls (CTR: C-1 and C-2) and laforin- (Laforin-: L-1 and L-2) and malin-deficient (Malin-: M-1) were incubated for 18 h in full medium without (-CCCP) or with (+CCCP) 10 μM CCCP, as indicated. For the last 2 h of treatment, fibroblasts were incubated in full (A) or in KH (B) media containing lysosomal inhibitors (100 μM leupeptin and 1 μM pepstatin A). Representative fluorescence microscopy images of control (C-1) and LD (L-2 and M-1) fibroblasts incubated with anti-LC3 are shown. Bar: 10 μm. The histograms (C and D, for full and KH media, respectively) show the mean (value indicated above the corresponding bar) and standard deviation of the number of LC3 puncta, quantified in 50 cells from control (CTR, red bars), laforin-deficient (Laforin-, green bars) and malin-deficient (Malin-, blue bars) fibroblasts in two different experiments (indicated in the histograms). Differences were found to be statistically significant at *P
Figure Legend Snippet: Number of autophagic vacuoles in control and in LD fibroblasts in the presence or absence of CCCP Fibroblasts from controls (CTR: C-1 and C-2) and laforin- (Laforin-: L-1 and L-2) and malin-deficient (Malin-: M-1) were incubated for 18 h in full medium without (-CCCP) or with (+CCCP) 10 μM CCCP, as indicated. For the last 2 h of treatment, fibroblasts were incubated in full (A) or in KH (B) media containing lysosomal inhibitors (100 μM leupeptin and 1 μM pepstatin A). Representative fluorescence microscopy images of control (C-1) and LD (L-2 and M-1) fibroblasts incubated with anti-LC3 are shown. Bar: 10 μm. The histograms (C and D, for full and KH media, respectively) show the mean (value indicated above the corresponding bar) and standard deviation of the number of LC3 puncta, quantified in 50 cells from control (CTR, red bars), laforin-deficient (Laforin-, green bars) and malin-deficient (Malin-, blue bars) fibroblasts in two different experiments (indicated in the histograms). Differences were found to be statistically significant at *P

Techniques Used: Incubation, Fluorescence, Microscopy, Standard Deviation

Colocalization of mitochondrial and lysosomal markers in control and LD fibroblasts in the presence or absence of CCCP Fibroblasts from controls and laforin- and malin-deficient patients were stained with 100 nM MitoTracker Red for 30 min at 37 °C, washed and then treated or not with 10 μM CCCP in full medium for 18 h. Lysosomal inhibitors (100 μM leupeptin and 1 μM pepstatin A) were added for the last 2 h of the 18 h incubation period. Then, fibroblasts were immunostained with anti-LAMP2. Representative fluorescence microscopy images of control (C-1, A and B) and LD (L-2, C and D) fibroblasts, treated (B and D) or not (A and C) with CCCP. Two-dimensional scatter plots corresponding to red (mitochondria) and green (lysosomes) pixel intensities from B and D are shown on their rights. Bar: 10 μm. The histogram (E) shows the quantification (mean and standard deviation as percentage of the highest control value) of MitoTracker Red and LAMP2 colocalization after CCCP treatment in the different cells using MetaMorph software (CTR: mean values from C-1 and C2 cells; Laforin-: mean values from L-1 and L-2 cells; Malin-: M-1 cells). The colocalization of mitochondria with lysosomes is reduced in LD fibroblasts (Laforin-, green bars and Malin-, blue bars) compared to controls (CTR, red bars). Experiments were performed in duplicate, analysing 40 cells for each cell line. Differences were found to be statistically significant at ***P
Figure Legend Snippet: Colocalization of mitochondrial and lysosomal markers in control and LD fibroblasts in the presence or absence of CCCP Fibroblasts from controls and laforin- and malin-deficient patients were stained with 100 nM MitoTracker Red for 30 min at 37 °C, washed and then treated or not with 10 μM CCCP in full medium for 18 h. Lysosomal inhibitors (100 μM leupeptin and 1 μM pepstatin A) were added for the last 2 h of the 18 h incubation period. Then, fibroblasts were immunostained with anti-LAMP2. Representative fluorescence microscopy images of control (C-1, A and B) and LD (L-2, C and D) fibroblasts, treated (B and D) or not (A and C) with CCCP. Two-dimensional scatter plots corresponding to red (mitochondria) and green (lysosomes) pixel intensities from B and D are shown on their rights. Bar: 10 μm. The histogram (E) shows the quantification (mean and standard deviation as percentage of the highest control value) of MitoTracker Red and LAMP2 colocalization after CCCP treatment in the different cells using MetaMorph software (CTR: mean values from C-1 and C2 cells; Laforin-: mean values from L-1 and L-2 cells; Malin-: M-1 cells). The colocalization of mitochondria with lysosomes is reduced in LD fibroblasts (Laforin-, green bars and Malin-, blue bars) compared to controls (CTR, red bars). Experiments were performed in duplicate, analysing 40 cells for each cell line. Differences were found to be statistically significant at ***P

Techniques Used: Staining, Incubation, Fluorescence, Microscopy, Standard Deviation, Software

LC3-II levels in control and in LD fibroblasts in the presence or absence of CCCP Fibroblasts from controls (CTR: C-1 and C-2) and patients with LD, laforin- (Laforin-: L-1 and L-2) and malin-deficient (Malin-: M-1) were incubated for 18 h in full medium without (-CCCP) or with (+CCCP) 10 μM CCCP, as indicated. For the last 2 h of the CCCP treatment, fibroblasts were incubated in full or in KH media containing lysosomal inhibitors (20 mM ammonium chloride and 100 μM leupeptin). Cellular extracts (75 μg protein) were analysed by Western blot using anti-LC3 and anti-actin. Representative immunoblots for full (A) and KH (B) media are shown. The ratio of LC3-II to actin levels in control (CTR, red bars), laforin-deficient (Laforin-, green bars) and malin-deficient (Malin-, blue bars) fibroblasts was determined densitometrically and normalized in percentage to the highest control value (C and D, for full and KH media, respectively). All values are means and standard deviations of five independent experiments. Differences were found to be statistically significant at *P
Figure Legend Snippet: LC3-II levels in control and in LD fibroblasts in the presence or absence of CCCP Fibroblasts from controls (CTR: C-1 and C-2) and patients with LD, laforin- (Laforin-: L-1 and L-2) and malin-deficient (Malin-: M-1) were incubated for 18 h in full medium without (-CCCP) or with (+CCCP) 10 μM CCCP, as indicated. For the last 2 h of the CCCP treatment, fibroblasts were incubated in full or in KH media containing lysosomal inhibitors (20 mM ammonium chloride and 100 μM leupeptin). Cellular extracts (75 μg protein) were analysed by Western blot using anti-LC3 and anti-actin. Representative immunoblots for full (A) and KH (B) media are shown. The ratio of LC3-II to actin levels in control (CTR, red bars), laforin-deficient (Laforin-, green bars) and malin-deficient (Malin-, blue bars) fibroblasts was determined densitometrically and normalized in percentage to the highest control value (C and D, for full and KH media, respectively). All values are means and standard deviations of five independent experiments. Differences were found to be statistically significant at *P

Techniques Used: Incubation, Western Blot

Effect of oligomycin plus antimycin A treatment on the LC3-II levels in control and in LD fibroblasts Control (C-1 and C-2), laforin-deficient (L-1 and L-2) and malin-deficient (M-1) fibroblasts were incubated in full medium without (-OA) or with (+OA) 10 μM oligomycin plus 1 μM antimycin A for 18 h. For the last 2 h of treatment, fibroblasts were incubated in full (A) or in KH (B) media with lysosomal inhibitors (20 mM ammonium chloride and 100 μM leupeptin). Cellular extracts (75 μg protein) from the different samples were analysed by Western blot using anti-LC3 and anti-actin antibodies. Representative immunoblots are shown. The intensity of the bands was assessed by densitometry and the LC3-II values were normalized to those of actin in the same lanes (C and D, for full and KH media, respectively) in control (CTR, red bars), laforin-deficient (Laforin-, green bars) and malin-deficient (Malin-, blue bars) fibroblasts. In the histograms, the results are expressed as percentage of the respective highest control value. All values correspond to the means and standard deviations of five independent experiments. Differences were found to be statistically significant at **P
Figure Legend Snippet: Effect of oligomycin plus antimycin A treatment on the LC3-II levels in control and in LD fibroblasts Control (C-1 and C-2), laforin-deficient (L-1 and L-2) and malin-deficient (M-1) fibroblasts were incubated in full medium without (-OA) or with (+OA) 10 μM oligomycin plus 1 μM antimycin A for 18 h. For the last 2 h of treatment, fibroblasts were incubated in full (A) or in KH (B) media with lysosomal inhibitors (20 mM ammonium chloride and 100 μM leupeptin). Cellular extracts (75 μg protein) from the different samples were analysed by Western blot using anti-LC3 and anti-actin antibodies. Representative immunoblots are shown. The intensity of the bands was assessed by densitometry and the LC3-II values were normalized to those of actin in the same lanes (C and D, for full and KH media, respectively) in control (CTR, red bars), laforin-deficient (Laforin-, green bars) and malin-deficient (Malin-, blue bars) fibroblasts. In the histograms, the results are expressed as percentage of the respective highest control value. All values correspond to the means and standard deviations of five independent experiments. Differences were found to be statistically significant at **P

Techniques Used: Incubation, Western Blot

Colocalization of mitochondrial and lysosomal markers in control and LD fibroblasts in the presence or absence of oligomycin plus antimycin A Fibroblasts from controls (C-1 and C-2, A and B, respectively), and laforin- (L-1 and L-2, C and D, respectively) and malin-deficient (M-1, E) patients were treated with 10 μM oligomycin plus 1 μM antimycin A in full medium for 18 h. Lysosomal inhibitors (100 μM leupeptin and 1 μM pepstatin A) were added for the last 2 h of the 18 h incubation period. Then, fibroblasts were immunostained with anti-LAMP2 (green) and anti-TOM20 (red). Representative fluorescence microscopy images are shown. Bar: 10 μm. The histogram (F) shows the quantification (mean and standard deviation as percentage of the highest control value) of TOM20 and LAMP2 colocalization after oligomycin plus antimycin A treatment in the different cells using MetaMorph software. The colocalization of mitochondria with lysosomes is reduced in LD fibroblasts (Laforin-, green bars, and Malin-, blue bars) compared to controls (CTR, red bars). Experiments were performed in duplicate, analysing 40 cells for each cell line. Differences were found to be statistically significant at ***P
Figure Legend Snippet: Colocalization of mitochondrial and lysosomal markers in control and LD fibroblasts in the presence or absence of oligomycin plus antimycin A Fibroblasts from controls (C-1 and C-2, A and B, respectively), and laforin- (L-1 and L-2, C and D, respectively) and malin-deficient (M-1, E) patients were treated with 10 μM oligomycin plus 1 μM antimycin A in full medium for 18 h. Lysosomal inhibitors (100 μM leupeptin and 1 μM pepstatin A) were added for the last 2 h of the 18 h incubation period. Then, fibroblasts were immunostained with anti-LAMP2 (green) and anti-TOM20 (red). Representative fluorescence microscopy images are shown. Bar: 10 μm. The histogram (F) shows the quantification (mean and standard deviation as percentage of the highest control value) of TOM20 and LAMP2 colocalization after oligomycin plus antimycin A treatment in the different cells using MetaMorph software. The colocalization of mitochondria with lysosomes is reduced in LD fibroblasts (Laforin-, green bars, and Malin-, blue bars) compared to controls (CTR, red bars). Experiments were performed in duplicate, analysing 40 cells for each cell line. Differences were found to be statistically significant at ***P

Techniques Used: Incubation, Fluorescence, Microscopy, Standard Deviation, Software

11) Product Images from "IRS-1 acts as an endocytic regulator of IGF-I receptor to facilitate sustained IGF signaling"

Article Title: IRS-1 acts as an endocytic regulator of IGF-I receptor to facilitate sustained IGF signaling

Journal: eLife

doi: 10.7554/eLife.32893

IRS-1 inhibits the targeting of IGF-IR into lysosomes. ( A ) L6 cells stably expressing IGF-IR-EGFP were transfected with non-targeting or IRS-1 siRNA. The cells were stimulated with IGF-I in the presence of leupeptin and pepstatin A for 1 hr. Prior to fixation, they were incubated with LysoTracker (magenta) for staining lysosomes. The fixed cells were immunostained with anti-phospho-IGF-IR antibody (green), and the images were obtained by confocal microscopy. Insets show representative regions at higher magnification. Bar, 10 μm. ( B ) Quantification of colocalization between phospho-IGF-IR and LysoTracker in ( A ). The mean ± SD is shown (n > 20 cells). Differences were analyzed by ANOVA and the Tukey post hoc test. *p
Figure Legend Snippet: IRS-1 inhibits the targeting of IGF-IR into lysosomes. ( A ) L6 cells stably expressing IGF-IR-EGFP were transfected with non-targeting or IRS-1 siRNA. The cells were stimulated with IGF-I in the presence of leupeptin and pepstatin A for 1 hr. Prior to fixation, they were incubated with LysoTracker (magenta) for staining lysosomes. The fixed cells were immunostained with anti-phospho-IGF-IR antibody (green), and the images were obtained by confocal microscopy. Insets show representative regions at higher magnification. Bar, 10 μm. ( B ) Quantification of colocalization between phospho-IGF-IR and LysoTracker in ( A ). The mean ± SD is shown (n > 20 cells). Differences were analyzed by ANOVA and the Tukey post hoc test. *p

Techniques Used: Stable Transfection, Expressing, Transfection, Incubation, Staining, Confocal Microscopy

AP2, but not AP1, is required for the targeting of activated IGF-IR from the plasma membrane into lysosomes. ( A ) L6 cells were transfected with non-targeting or μ1 siRNA followed by IGF-I stimulation for the indicated time. Changes in phospho-IGF-IR were analyzed by immunoprecipitation and immunoblotting with the indicated antibodies. ( B ) L6 cells stably expressing IGF-IR-EGFP were transfected with non-targeting or μ2 siRNA. The cells were stimulated with IGF-I in the presence of leupeptin and pepstatin A for the indicated time. Prior to fixation, they were labeled with LysoTracker (magenta). The fixed cells were immunostained with anti-phospho-IGF-IR antibody (green), and the images were obtained by confocal microscopy. Insets show representative regions at higher magnification. Arrows are representative showing colocalization between phospho-IGF-IR and LysoTracker. Bar, 10 μm. ( C ) Quantification of colocalization between phospho-IGF-IR and LysoTracker in ( B ). The mean ±SD is shown (n > 20 cells). Differences were analyzed by ANOVA and the Tukey post hoc test. *p
Figure Legend Snippet: AP2, but not AP1, is required for the targeting of activated IGF-IR from the plasma membrane into lysosomes. ( A ) L6 cells were transfected with non-targeting or μ1 siRNA followed by IGF-I stimulation for the indicated time. Changes in phospho-IGF-IR were analyzed by immunoprecipitation and immunoblotting with the indicated antibodies. ( B ) L6 cells stably expressing IGF-IR-EGFP were transfected with non-targeting or μ2 siRNA. The cells were stimulated with IGF-I in the presence of leupeptin and pepstatin A for the indicated time. Prior to fixation, they were labeled with LysoTracker (magenta). The fixed cells were immunostained with anti-phospho-IGF-IR antibody (green), and the images were obtained by confocal microscopy. Insets show representative regions at higher magnification. Arrows are representative showing colocalization between phospho-IGF-IR and LysoTracker. Bar, 10 μm. ( C ) Quantification of colocalization between phospho-IGF-IR and LysoTracker in ( B ). The mean ±SD is shown (n > 20 cells). Differences were analyzed by ANOVA and the Tukey post hoc test. *p

Techniques Used: Transfection, Immunoprecipitation, Stable Transfection, Expressing, Labeling, Confocal Microscopy

12) Product Images from "Resveratrol directly targets DDX5 resulting in suppression of the mTORC1 pathway in prostate cancer"

Article Title: Resveratrol directly targets DDX5 resulting in suppression of the mTORC1 pathway in prostate cancer

Journal: Cell Death & Disease

doi: 10.1038/cddis.2016.114

Treatment with resveratrol degrades DDX5 protein. ( a and b ) PC-3 cells were treated with the indicated concentrations of resveratrol for 24 h ( a ) or every 24 h for 72 h ( b ). DDX5 protein was detected by western blotting. ( c ) Quantitative reverse transcriptase–PCR analysis of DDX5 mRNA in PC-3 cells treated with resveratrol for 24 h. Data are means±S.D. ( n =3). ( d ) PC-3 cells were incubated for the indicated times with or without 50 μ M resveratrol and/or 20 μ M cycloheximide. DDX5 protein was detected by western blotting. ( e ) PC-3 cells were incubated for 24 h with or without 20 μ M lactacystin, 1 μ M bafilomycin A1, 100 μ M leupeptin, 100 μ M antipain, 1 μ M pepstatin A, or 10 mM EDTA and/or 50 μ M resveratrol. DDX5 protein was detected by western blotting
Figure Legend Snippet: Treatment with resveratrol degrades DDX5 protein. ( a and b ) PC-3 cells were treated with the indicated concentrations of resveratrol for 24 h ( a ) or every 24 h for 72 h ( b ). DDX5 protein was detected by western blotting. ( c ) Quantitative reverse transcriptase–PCR analysis of DDX5 mRNA in PC-3 cells treated with resveratrol for 24 h. Data are means±S.D. ( n =3). ( d ) PC-3 cells were incubated for the indicated times with or without 50 μ M resveratrol and/or 20 μ M cycloheximide. DDX5 protein was detected by western blotting. ( e ) PC-3 cells were incubated for 24 h with or without 20 μ M lactacystin, 1 μ M bafilomycin A1, 100 μ M leupeptin, 100 μ M antipain, 1 μ M pepstatin A, or 10 mM EDTA and/or 50 μ M resveratrol. DDX5 protein was detected by western blotting

Techniques Used: Western Blot, Polymerase Chain Reaction, Incubation

13) Product Images from "Effects of a non-cyclodextrin cyclic carbohydrate on mouse melanoma cells: Characterization of a new type of hypopigmenting sugar"

Article Title: Effects of a non-cyclodextrin cyclic carbohydrate on mouse melanoma cells: Characterization of a new type of hypopigmenting sugar

Journal: PLoS ONE

doi: 10.1371/journal.pone.0186640

CNN induced drastic changes in the immunostaining pattern of lysosomes. (A) Expression of the lysosomal marker protein LAMP-1 (green) was detected in B16 cells treated with 50 mM CNN for 0, 1, 4, and 7 days, together with nuclear staining (blue). (B) Two-color immunostaining analysis of tyrosinase and LAMP-1. B16 cells were untreated (upper panels) and treated (lower panels) with 50 mM CNN for 4 days. Tyrosinase (red) and LAMP-1 (green) were detected with anti-tyrosinase and anti-LAMP-1 antibodies, respectively. Merged three-color images (red [tyrosinase], green [LAMP-1], and blue [nuclei]) and DIC images (far right) are also shown. (C) Influence of lysosomal inhibitors on the depigmenting effect of CNN. B16 cells were treated with CNN (50 mM) in the presence of the lysosomal inhibitors leupeptin/pepstatin (L/P, at 2 and 5 mM) or NH 4 Cl (1 mM) for 4 days. Following treatment, levels of melanin and total protein were measured. P > 0.05 denotes the absence of statistical significance between the indicated pair of groups, as analyzed by one-way ANOVA and Tukey-Kramer post hoc testing. Data are representative results of at least two independent experiments.
Figure Legend Snippet: CNN induced drastic changes in the immunostaining pattern of lysosomes. (A) Expression of the lysosomal marker protein LAMP-1 (green) was detected in B16 cells treated with 50 mM CNN for 0, 1, 4, and 7 days, together with nuclear staining (blue). (B) Two-color immunostaining analysis of tyrosinase and LAMP-1. B16 cells were untreated (upper panels) and treated (lower panels) with 50 mM CNN for 4 days. Tyrosinase (red) and LAMP-1 (green) were detected with anti-tyrosinase and anti-LAMP-1 antibodies, respectively. Merged three-color images (red [tyrosinase], green [LAMP-1], and blue [nuclei]) and DIC images (far right) are also shown. (C) Influence of lysosomal inhibitors on the depigmenting effect of CNN. B16 cells were treated with CNN (50 mM) in the presence of the lysosomal inhibitors leupeptin/pepstatin (L/P, at 2 and 5 mM) or NH 4 Cl (1 mM) for 4 days. Following treatment, levels of melanin and total protein were measured. P > 0.05 denotes the absence of statistical significance between the indicated pair of groups, as analyzed by one-way ANOVA and Tukey-Kramer post hoc testing. Data are representative results of at least two independent experiments.

Techniques Used: Immunostaining, Expressing, Marker, Staining

Related Articles

Protease Inhibitor:

Article Title: Autophagic activity measured in whole rat hepatocytes as the accumulation of a novel BHMT fragment (p10), generated in amphisomes by the asparaginyl proteinase, legumain
Article Snippet: .. Leupeptin (4041) was obtained from the Peptide Institute; aprotinin (A2132), bestatin-HCl (A2137), E-64 (A2157) and pepstatin A (A2205) from AppliChem; okadaic acid (ALX-350-010) from Alexis and the protease inhibitor cocktail set III (539134) from Calbiochem. .. 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF; Pefabloc; 76307), ammonium bicarbonate (A6141), ammonium chloride (A4514), l-asparagine monohydrate (A8381), bromophenol blue (B5525), CAPS (C2632), CHAPS (C9426), α-cyano-4-hydroxycinnamic acid (C2020), dimethylsulfoxide (41640), DL-dithiothreitol (D9163), EDTA (ED3SS), formaldehyde (F1635), glycerol (G5150), glycine (G7126), HEPES (H3375), 3-methyladenine (M9281), β-nicotinamide adenine dinucleotide (NAD; N8129), potassium phosphate monobasic (P0662), propylamine (240958), sodium deoxycholate monohydrate (D5670), sodium fluoride (S6521), sodium orthovanadate (S6508), sodium pyrophosphate (S6422), sodium sulfate (S9627), TEMED (T9281), TES (T1375), thiourea (T8656), trifluoroacetic acid (T6508), tricine (T0377), Trizma base (T1503), Tween 20 (P1379) and vinblastine (V1377) were from Sigma-Aldrich.

other:

Article Title: The Amino Acid Specificity for Activation of Phenylalanine Hydroxylase Matches the Specificity for Stabilization of Regulatory Domain Dimers
Article Snippet: Leupeptin and pepstatin A were from Peptide Institute, Inc. (Osaka, Japan). l -Norleucine was purchased from MP Biomedicals, Inc. (Solon, OH).

Article Title: Minus end–directed motor KIFC3 suppresses E-cadherin degradation by recruiting USP47 to adherens junctions
Article Snippet: Leupeptin was purchased from Peptide Institute (Osaka, Japan).

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    Peptide Institute leupeptin
    Production of the 90-kDa E-cadherin fragment depends on proteasome and Hakai. (A) Effects of proteasome inhibitors on 90-kDa fragment production. Caco-2 cells were transfected with control or KIFC3-specific siRNA. At 7 h after transfection, cells were passaged. After 24 h, dimethyl sulfoxide, 1 μM MG132, 10 μM lactacystin, or 100 nM epoxomycin was added to culture medium. At 16 h later, cells were harvested and subjected to Western blotting assay using the antibodies indicated. (B) Effects of Hakai depletion on 90-kDa fragment production. At 48 h after transfection of cells with indicated siRNAs, cells were harvested and subjected to Western blotting assay using the antibodies indicated. Knockdown efficiency for each molecule is shown on the far right. (C) Effects of proteasome and lysosome inhibitors on 90-kDa fragment production. Cells were treated with siRNAs and subsequently with 10 mM NH 4 Cl and 21 μM <t>leupeptin</t> with or without 100 nM epoxomycin, according to the same protocol as in A.
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    Production of the 90-kDa E-cadherin fragment depends on proteasome and Hakai. (A) Effects of proteasome inhibitors on 90-kDa fragment production. Caco-2 cells were transfected with control or KIFC3-specific siRNA. At 7 h after transfection, cells were passaged. After 24 h, dimethyl sulfoxide, 1 μM MG132, 10 μM lactacystin, or 100 nM epoxomycin was added to culture medium. At 16 h later, cells were harvested and subjected to Western blotting assay using the antibodies indicated. (B) Effects of Hakai depletion on 90-kDa fragment production. At 48 h after transfection of cells with indicated siRNAs, cells were harvested and subjected to Western blotting assay using the antibodies indicated. Knockdown efficiency for each molecule is shown on the far right. (C) Effects of proteasome and lysosome inhibitors on 90-kDa fragment production. Cells were treated with siRNAs and subsequently with 10 mM NH 4 Cl and 21 μM leupeptin with or without 100 nM epoxomycin, according to the same protocol as in A.

    Journal: Molecular Biology of the Cell

    Article Title: Minus end–directed motor KIFC3 suppresses E-cadherin degradation by recruiting USP47 to adherens junctions

    doi: 10.1091/mbc.E14-07-1245

    Figure Lengend Snippet: Production of the 90-kDa E-cadherin fragment depends on proteasome and Hakai. (A) Effects of proteasome inhibitors on 90-kDa fragment production. Caco-2 cells were transfected with control or KIFC3-specific siRNA. At 7 h after transfection, cells were passaged. After 24 h, dimethyl sulfoxide, 1 μM MG132, 10 μM lactacystin, or 100 nM epoxomycin was added to culture medium. At 16 h later, cells were harvested and subjected to Western blotting assay using the antibodies indicated. (B) Effects of Hakai depletion on 90-kDa fragment production. At 48 h after transfection of cells with indicated siRNAs, cells were harvested and subjected to Western blotting assay using the antibodies indicated. Knockdown efficiency for each molecule is shown on the far right. (C) Effects of proteasome and lysosome inhibitors on 90-kDa fragment production. Cells were treated with siRNAs and subsequently with 10 mM NH 4 Cl and 21 μM leupeptin with or without 100 nM epoxomycin, according to the same protocol as in A.

    Article Snippet: Leupeptin was purchased from Peptide Institute (Osaka, Japan).

    Techniques: Transfection, Western Blot

    Inhibition of the ubiquitin-proteasome pathway and activation of autophagy by PTEN in U87MG cells are independent of mTOR. U87MG cells expressing WT-PTEN or C124S-PTEN ( A ) or WT-PTEN or G129E-PTEN and mock-treated ( B ) were incubated for 18 h with doxycycline and in the last 2 h the following inhibitors were added as indicated: rapamycin (RAP, 200 mM, mTOR inhibitor), KT5720 (25 µM, PKA inhibitor), PD98059 (10 µM, ERK1/2 inhibitor) and SB203580 (10 µM, p38 inhibitor). Before collecting the cells, proteasome (50 µM MG132, A ) and lysosomal (100 µM leupeptin and 20 mM NH 4 Cl, B) inhibitors were also added for 1 h. Total lysates were analyzed by SDS-PAGE and Western blot with antibodies that recognize ubiquitinated proteins (FK1) ( A ), LC3 ( B ) and, as a loading control actin. Molecular weight markers are indicated on the right and in B the position of LC3-I, and LC3-II bands are also shown. The histograms on the right show means ± SD of the densitometric measurements from three different experiments and are expressed as amounts of ubiquitinated proteins (ubiq. prot.) normalized to the levels of actin ( A ) or as LC3II/actin ratios ( B ). Stars indicate statistically significant differences from the values without the corresponding inhibitor treatment at ** p

    Journal: PLoS ONE

    Article Title: PTEN Increases Autophagy and Inhibits the Ubiquitin-Proteasome Pathway in Glioma Cells Independently of its Lipid Phosphatase Activity

    doi: 10.1371/journal.pone.0083318

    Figure Lengend Snippet: Inhibition of the ubiquitin-proteasome pathway and activation of autophagy by PTEN in U87MG cells are independent of mTOR. U87MG cells expressing WT-PTEN or C124S-PTEN ( A ) or WT-PTEN or G129E-PTEN and mock-treated ( B ) were incubated for 18 h with doxycycline and in the last 2 h the following inhibitors were added as indicated: rapamycin (RAP, 200 mM, mTOR inhibitor), KT5720 (25 µM, PKA inhibitor), PD98059 (10 µM, ERK1/2 inhibitor) and SB203580 (10 µM, p38 inhibitor). Before collecting the cells, proteasome (50 µM MG132, A ) and lysosomal (100 µM leupeptin and 20 mM NH 4 Cl, B) inhibitors were also added for 1 h. Total lysates were analyzed by SDS-PAGE and Western blot with antibodies that recognize ubiquitinated proteins (FK1) ( A ), LC3 ( B ) and, as a loading control actin. Molecular weight markers are indicated on the right and in B the position of LC3-I, and LC3-II bands are also shown. The histograms on the right show means ± SD of the densitometric measurements from three different experiments and are expressed as amounts of ubiquitinated proteins (ubiq. prot.) normalized to the levels of actin ( A ) or as LC3II/actin ratios ( B ). Stars indicate statistically significant differences from the values without the corresponding inhibitor treatment at ** p

    Article Snippet: Leupeptin and N-Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (N-Suc-LLVY-MCA) were from Peptide Institute, Inc. and LysoTracker Red from Molecular Probes-Invitrogen Life Technologies.

    Techniques: Inhibition, Activation Assay, Expressing, Incubation, SDS Page, Western Blot, Molecular Weight

    PTEN expression in U87MG cells increases the formation of autophagosomes. A ) U87MG cells expressing WT-PTEN, C124S-PTEN and G129E-PTEN or mock-treated were incubated in high (H) or low (L) proteolysis media (see Materials and Methods) for 2 h in the presence of lysosomal inhibitors (100 µM leupeptin and 20 mM NH 4 Cl). Extracts (75 µg protein) were analyzed by SDS-PAGE and immunoblot, with low and high exposure (exp.), using an antibody against LC3 and, as a loading control, an antibody that recognizes actin. A representative experiment is shown. The position of LC3-I and LC3-II bands are indicated on the left and molecular weight markers are indicated on the right. The histogram on the right shows the means ± SD of the densitometric analysis of the LC3-II/actin ratios from five different experiments. Stars indicate statistically significant differences from the corresponding (high or low proteolysis conditions) values in mock-treated cells at ** p

    Journal: PLoS ONE

    Article Title: PTEN Increases Autophagy and Inhibits the Ubiquitin-Proteasome Pathway in Glioma Cells Independently of its Lipid Phosphatase Activity

    doi: 10.1371/journal.pone.0083318

    Figure Lengend Snippet: PTEN expression in U87MG cells increases the formation of autophagosomes. A ) U87MG cells expressing WT-PTEN, C124S-PTEN and G129E-PTEN or mock-treated were incubated in high (H) or low (L) proteolysis media (see Materials and Methods) for 2 h in the presence of lysosomal inhibitors (100 µM leupeptin and 20 mM NH 4 Cl). Extracts (75 µg protein) were analyzed by SDS-PAGE and immunoblot, with low and high exposure (exp.), using an antibody against LC3 and, as a loading control, an antibody that recognizes actin. A representative experiment is shown. The position of LC3-I and LC3-II bands are indicated on the left and molecular weight markers are indicated on the right. The histogram on the right shows the means ± SD of the densitometric analysis of the LC3-II/actin ratios from five different experiments. Stars indicate statistically significant differences from the corresponding (high or low proteolysis conditions) values in mock-treated cells at ** p

    Article Snippet: Leupeptin and N-Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (N-Suc-LLVY-MCA) were from Peptide Institute, Inc. and LysoTracker Red from Molecular Probes-Invitrogen Life Technologies.

    Techniques: Expressing, Incubation, SDS Page, Molecular Weight

    PTEN expression in U87MG cells increases the formation of autophagosomes under various proteolysis conditions. U87MG cells expressing WT-PTEN, G129E-PTEN or C124S-PTEN ( A ) and mock-treated U87MG cells or expressing WT-PTEN or C124S-PTEN ( B ) were incubated under high (KH), intermediate (Ins or EAA)( A and B ) and low (Ins/EAA)(only in A ) proteolysis conditions for 2 h as in Figure 5C in the presence of lysosomal inhibitors (100 µM leupeptin and 20 mM NH 4 Cl). Extracts (75 µg protein) were analyzed by SDS-PAGE and immunoblot with antibodies that recognize LC3 and, as a loading control, actin. The position of LC3-I and LC3-II bands are indicated on the left and molecular weight markers are indicated on the right. The Western blots on the left show representative experiments and the histograms on the right show the means ± SD of the densitometric analysis of the LC3-II/ratios from five different experiments. Stars indicate statistically significant differences from the corresponding (high, intermediate or low proteolysis conditions) values in the cells expressing the C124S PTEN mutant ( A ) and mock-treated cells ( B ) at ** p

    Journal: PLoS ONE

    Article Title: PTEN Increases Autophagy and Inhibits the Ubiquitin-Proteasome Pathway in Glioma Cells Independently of its Lipid Phosphatase Activity

    doi: 10.1371/journal.pone.0083318

    Figure Lengend Snippet: PTEN expression in U87MG cells increases the formation of autophagosomes under various proteolysis conditions. U87MG cells expressing WT-PTEN, G129E-PTEN or C124S-PTEN ( A ) and mock-treated U87MG cells or expressing WT-PTEN or C124S-PTEN ( B ) were incubated under high (KH), intermediate (Ins or EAA)( A and B ) and low (Ins/EAA)(only in A ) proteolysis conditions for 2 h as in Figure 5C in the presence of lysosomal inhibitors (100 µM leupeptin and 20 mM NH 4 Cl). Extracts (75 µg protein) were analyzed by SDS-PAGE and immunoblot with antibodies that recognize LC3 and, as a loading control, actin. The position of LC3-I and LC3-II bands are indicated on the left and molecular weight markers are indicated on the right. The Western blots on the left show representative experiments and the histograms on the right show the means ± SD of the densitometric analysis of the LC3-II/ratios from five different experiments. Stars indicate statistically significant differences from the corresponding (high, intermediate or low proteolysis conditions) values in the cells expressing the C124S PTEN mutant ( A ) and mock-treated cells ( B ) at ** p

    Article Snippet: Leupeptin and N-Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (N-Suc-LLVY-MCA) were from Peptide Institute, Inc. and LysoTracker Red from Molecular Probes-Invitrogen Life Technologies.

    Techniques: Expressing, Incubation, SDS Page, Molecular Weight, Western Blot, Mutagenesis

    Gel filtration of RDTPH1 C64S. Conditions: 100 mM NaCl, 1 µM pepstatin A, 1 µM leupeptin, 50 mM phosphate, pH 8.0, 23 °C. Inset: elution volumes of the standard proteins aprotinin (6,500 Da), ribonuclease A (13,700 Da), carbonic

    Journal: Biochemical and biophysical research communications

    Article Title: The Regulatory Domain of Human Tryptophan Hydroxylase 1 Forms a Stable Dimer

    doi: 10.1016/j.bbrc.2016.05.144

    Figure Lengend Snippet: Gel filtration of RDTPH1 C64S. Conditions: 100 mM NaCl, 1 µM pepstatin A, 1 µM leupeptin, 50 mM phosphate, pH 8.0, 23 °C. Inset: elution volumes of the standard proteins aprotinin (6,500 Da), ribonuclease A (13,700 Da), carbonic

    Article Snippet: Leupeptin and pepstatin A were from Peptide Institute, Inc (Osaka, Japan).

    Techniques: Filtration

    Effect of activation and inhibition of p38 MAPK on endocytosis and intracellular sorting of FGF1. (A) NIH 3T3 cells were incubated with SB203580, SB202474, anisomycin, or PD169316 and increasing concentrations of 125 I-labeled FGF1 for 3 h at 4°C. The cells were washed with 1 M NaCl and lysed, and the solubilized radioactivity, representing the cell surface FGFR-bound 125 I-FGF1, was measured (reported as counts per minute [CPM]). (B) HeLa cells transfected with FGFR1 or FGFR4 were incubated with Cy3-FGF1 and heparin in the presence or absence of SB203580 (10 μM) or anisomycin (10 μM) for 2 h at 37°C in DMEM with 0.3 mM leupeptin. The cells were fixed, stained for LAMP-1 (Cy2), and examined by confocal microscopy. Bar, 5 μm. Images shown are merged images (red plus green) where a section of each image has been magnified and shown to the left as red, green, and merged images separately. The proportion of Cy3-positive structures that colocalized with Cy2-positive structures (indicated by yellow in the merged images) was calculated and presented as the mean from 15 cells in each case. Error bars indicate the standard deviations. (C) NIH 3T3 cells were preincubated with or without 20 μM anisomycin for 15 min at 37°C and then incubated with 35 S-FGF1 and heparin at 4°C for 2 h. The cells were washed with 1 M NaCl and lysed to measure surface-bound FGF1 or, to measure endocytosis, incubated further for 0, 15, 30, or 60 min at 37°C in the presence and absence of 20 μM anisomycin. 35 S-FGF1 was extracted from cell lysates and subjected to SDS-PAGE and fluorography. *, partially degraded FGF1.

    Journal: Molecular and Cellular Biology

    Article Title: Phosphorylation of Fibroblast Growth Factor (FGF) Receptor 1 at Ser777 by p38 Mitogen-Activated Protein Kinase Regulates Translocation of Exogenous FGF1 to the Cytosol and Nucleus

    doi: 10.1128/MCB.02117-07

    Figure Lengend Snippet: Effect of activation and inhibition of p38 MAPK on endocytosis and intracellular sorting of FGF1. (A) NIH 3T3 cells were incubated with SB203580, SB202474, anisomycin, or PD169316 and increasing concentrations of 125 I-labeled FGF1 for 3 h at 4°C. The cells were washed with 1 M NaCl and lysed, and the solubilized radioactivity, representing the cell surface FGFR-bound 125 I-FGF1, was measured (reported as counts per minute [CPM]). (B) HeLa cells transfected with FGFR1 or FGFR4 were incubated with Cy3-FGF1 and heparin in the presence or absence of SB203580 (10 μM) or anisomycin (10 μM) for 2 h at 37°C in DMEM with 0.3 mM leupeptin. The cells were fixed, stained for LAMP-1 (Cy2), and examined by confocal microscopy. Bar, 5 μm. Images shown are merged images (red plus green) where a section of each image has been magnified and shown to the left as red, green, and merged images separately. The proportion of Cy3-positive structures that colocalized with Cy2-positive structures (indicated by yellow in the merged images) was calculated and presented as the mean from 15 cells in each case. Error bars indicate the standard deviations. (C) NIH 3T3 cells were preincubated with or without 20 μM anisomycin for 15 min at 37°C and then incubated with 35 S-FGF1 and heparin at 4°C for 2 h. The cells were washed with 1 M NaCl and lysed to measure surface-bound FGF1 or, to measure endocytosis, incubated further for 0, 15, 30, or 60 min at 37°C in the presence and absence of 20 μM anisomycin. 35 S-FGF1 was extracted from cell lysates and subjected to SDS-PAGE and fluorography. *, partially degraded FGF1.

    Article Snippet: Leupeptin was from the Peptide Institute.

    Techniques: Activation Assay, Inhibition, Incubation, Labeling, Radioactivity, Transfection, Staining, Confocal Microscopy, SDS Page