Equal amounts of proteins from U87-MG cell lysates (30 μg of proteins) were mixed with Laemmli solution, run in 4–15% polyacrylamide gels (Mini-PROTEAN® Precast Gels, Biorad, Hercules, CA, USA) using a mini-Protean Tetracell (Biorad, Hercules, CA, USA), and transferred onto nitrocellulose membranes (0.2 μm) using a Trans-Blot Turbo transfer system (Biorad) as previously described [33 (link)]. Immediately after, WB membranes were stained with 1 mM RuBP and total protein images acquired by ImageQuant LAS4010 (GE Health Care, Uppsala, Sweden). Subsequently, membranes were incubated with primary antibodies (dilution 1:1000). The following antibodies were purchased from Cell Signaling Technology, Beverly, MA, USA: TFEB (D207D) rabbit mAb (#37785), phosphorylated TFEB (E9S8N) rabbit mAb (pTFEBS211; #37681), mTOR (7C10) rabbit mAb (#2983), phosphorylated mTOR (D9C2) XP rabbit mAb (p-mTORS2448; #5536). Immunoblots were developed using the enhanced chemiluminescence (ECL) detection system. The chemiluminescent images were acquired using LAS4010 (GE Health Care Europe, Upsala, Sweden). Semiquantitative analysis of specific immunolabeled bands was performed using ImageQuant TL 7 software.
Las4010
Manufactured by GE Healthcare
Sourced in United States, Sweden, Japan
The LAS4010 is a laboratory equipment product offered by GE Healthcare. It is a precision instrument designed for the analysis and processing of samples in a laboratory setting. The core function of the LAS4010 is to provide accurate and reliable measurements and data processing capabilities to support various laboratory workflows and research activities.
Automatically generated - may contain errors
Lab products found in correlation
Ecl plus detection reagent, by Santa Cruz Biotechnology (7 mentions)
Mini protean tetra cell, by Bio-Rad (4 mentions)
Trans blot turbo transfer system, by Bio-Rad (4 mentions)
Hybond, by Cytiva (4 mentions)
Mini protean precast gel, by Bio-Rad (3 mentions)
Hybond membrane, by Cytiva (3 mentions)
Mab1501, by Merck Group (3 mentions)
Enhanced chemi luminescence (ecl), by PerkinElmer (3 mentions)
Anti goat igg conjugated to horseradish peroxidase, by Agilent Technologies (2 mentions)
Wb stripping solution ph2 3, by Nacalai Tesque (2 mentions)
Sc 7870, by Santa Cruz Biotechnology (2 mentions)
Sc 7939, by Santa Cruz Biotechnology (2 mentions)
Anti rabbit igg light chains conjugated to peroxidase, by Merck Group (2 mentions)
Imagequant las 4010, by GE Healthcare (1 mentions)
Imagequant tl, by GE Healthcare (1 mentions)
Immobilon, by Merck Group (1 mentions)
Er isolation kit, by Merck Group (1 mentions)
Bca kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
32 protocols using las4010
1
Western Blot Analysis of TFEB and mTOR Signaling
2
Streptavidin Pulldown of Yeast Proteins
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Streptavidin (50 µg in 5 µL; Prozyme) was incubated with 20–40 µL of labeled yeast whole-cell extract for 10 min at room temperature. Samples were then diluted 1:4 in H2O followed by addition of an equivalent volume of colorless Laemelli buffer prior to SDS-PAGE. The samples were not heat denatured prior to loading. Gels were subsequently imaged using a GE LAS4010 and SNAPAP tag fluorescence quantified with ImageQuant software.
3
Protein Extraction and Western Blot Analysis
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Total protein was extracted by sonication in radio-immunoprecipitation assay (RIPA) buffer(50 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 5 mM dithiothreitol, 10 mM NaF, protease inhibitor cocktail). 100 μg denaturedprotein was separated on an SDS-polyacrylamide gel and transferred to Hybond membrane (Amersham, Germany), which was then blocked overnight in 5% skimmed milk inTris-bufferedsaline with Tween 20 (TTBS, 10 mM Tris–HCl, 150 mM NaCl, 0.1% Tween 20). For immunoblotting, the membrane was incubated for 15 min with antibodies. The membrane was rinsed with TBST and incubated with anti-mouse, anti-rabbit or anti-goat IgG conjugated to horseradish peroxidase (DAKO, USA, 1:1000) for 15 min. All the incubations were performed in a microwave oven to allow intermittent irradiation. Bands were visualized with LAS4010 (GE Healthcare Life Science, USA) by ECL-Plus detection reagents (Santa Cruz, USA). Densitometric quantification of protein bands was performed with GAPDH as an internal control using Image J (NIH, USA).
4
Immunoprecipitation and Immunoblotting Analysis
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After washing the cultured cells with PBS, cells were lysed in lysis buffer containing 1% Nonidet P-40 (NP-40) and protease inhibitors for 20 min, and insoluble materials were removed by centrifugation. For immunoprecipitation, protein extracts were incubated with the appropriate Ab and protein A-sepharose beads (Thermo Fisher Scientific) at 4 °C for 6 h and immunoprecipitates were obtained by centrifugation. PDI-positive ER fraction was isolated with an ER Isolation Kit (ER0100, Sigma-Aldrich) following their protocol. Cell lysates (15 μg per each lane) or immunoprecipitated proteins were subjected to SDS–PAGE and electrophoretically transferred onto a polyvinylidene difluoride membrane (Immobilon®, Millipore, Bedford, MA). After blocking residual binding sites on the membrane with TBST blocking buffer (4% nonfat dry milk in Tris-buffered saline-Tween 20, 0.15 M NaCl, 0.01 M Tris-HCl pH 7.4, 0.05% Tween 20), immunoblotting was performed with appropriate primary and secondary Abs. Then, immunoreactive proteins were visualized by an enhanced chemiluminescence kit (LAS4010, GE Healthcare, Cleveland, OH), and densitometric analyses were carried out by Image Quant TL (GE Healthcare). The original images of immunoblots analyses were shown in Supplementary Fig. 9 and 9contnd.
5
Cytokine Profiling of Monocyte Secretome
6
Quantitative Western Blot Analysis
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Western blot was carried out as described previously30 (link). Cells were lysed in lysis buffer containing a protease inhibitor and a phosphatase inhibitor cocktail (Roche) for 30min. The supernatant was collected as whole cell lysates and protein concentration was quantified using the BCA Kit (Thermo Fisher Scientific). 25 to 50 μg denatured protein was separated by SDS-polyacrylamide gel followed by transferring onto PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked in 5% skim milk for 1 hr and then probed with primary antibodies at 4°C overnight. After washing three times by TBST, the blots were incubated with corresponding secondary antibody at 37 °C for 1 hr. Finally, the bands were visualized by ECL detection kit (Pierce, Rockford, IL, USA) using LAS 4010 (GE Healthcare Life Sciences, Little Chalfont, UK).
7
Western Blot Analysis of SIRT1 Expression
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A portion of the left ventricular tissue was lysed in RIPA buffer, and proteins were quantified with the Bradford assay (Bio-Rad, USA). Proteins (25 μg) were separated onto a precast 4-20% polyacrylamide gel (Mini-PROTEAN® TGX gel, Bio-Rad, USA) and transferred to PVDF membranes (Trans-Blot® Turbo™ PVDF Transfer packs, Bio-Rad, USA). Membranes were blocked with 5% bovine serum albumin (BSA) diluted in Tris-buffered saline (TBS, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% Tween 20. Primary antibodies against GAPDH (1 : 5000, Millipore) and SIRT1 (1 : 100, Santa Cruz, USA) were incubated overnight at 4°C. Then, membranes were incubated with secondary antibodies (1 : 5000, anti-mouse and anti-rabbit) for 2 h at room temperature. The immunoblot signal was visualized by using enhanced chemiluminescence substrate detection system (Luminata™ Forte Western HRP Substrate, Millipore, USA). The chemiluminescent images were acquired by LAS4010 (GE Healthcare Life Sciences, USA). Densitometry was undertaken using ImageJ software.
Data analysis. One-way ANOVA followed by Bonferroni's post hoc test was selected as statistical analysis, and the difference between groups was considered statistically different when p < 0.05.
Data analysis. One-way ANOVA followed by Bonferroni's post hoc test was selected as statistical analysis, and the difference between groups was considered statistically different when p < 0.05.
8
Protein Extraction and Western Blot Analysis
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Tissues and cells were subjected to protein extraction by homogenization or sonication in radioimmunoprecipitation lysis assay buffer. Denatured protein was separated on SDS-polyacrylamide gel and transferred to Hybond membrane, which was then blocked overnight in 5% skim milk in TBST. For immunobloting, the membrane was incubated for 15 min with ani-BTG1 (Proteintech) or −β-actin (Sigma) antibody. Then, it was rinsed by TBST and incubated with IgG conjugated to horseradish peroxidase (DAKO, 1:1000) for 15 min. All the incubations were performed in a microwave oven to allow intermittent irradiation as recommended by Li et al. [29 (link)]. Bands were visualized with LAS 4010 (GE healthcare Life Science) by ECL-Plus detection reagents (Santa Cruz). Densitometric quantification of target proteins was performed using Scion Image software.
9
Western Blot Analysis of TRPV5 and TRPV6
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Western blot analyses were carried out on protein extracts (P1 and S1) as previously described 18 (link),20 (link). Aliquots of 60 μg of proteins were mixed with SDS sample buffer (Laemmli solution) and heated at 100°C for 5 min. Proteins were run on 8% SDS-PAGE gels, and transferred onto nitrocellulose membranes (0.2 μm) by using 100 V for 30 min. (Tetra cell apparatus, Bio-Rad). Anti-TRPV5 and anti-TRPV6 goat polyclonal antibodies (K-17 and L-15, respectively, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) were used at dilution of 1:200. After incubation with appropriate secondary antibody, immunoreactions were visualized by using ECL detection system. The chemiluminescent images were acquired by LAS4010 (GE Health Care Europe, Uppsala, Sweden).
10
Rac1 Activity Measurement by Pulldown Assay
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Rac1 activity was measured by pulldown assay using the active Rac1 detection kit (Cell Signaling), according to the manufacturer’s instructions. Cells were seeded in six-well plates (5 × 105 cells per well) and maintained for 24 h in a CO2 incubator. Cells were washed with cold PBS and lysed. Protein lysates were centrifuged and the supernatant was collected in new tubes containing beads pre-coupled with GST–PAK1-PBD and incubated under rotation at 4 °C for 60 min. The beads were washed and the proteins bound to them were separated by SDS-PAGE. The amounts of active Rac1 were determined by immunoblot analysis. Signals were detected using a LAS4010 imaging system (GE Healthcare, Chicago, IL, USA).
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