For detection of TFE3, cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, washed with PBS containing 0.3% Triton X-100, incubated with an antibody against either TFE3 (Sigma, #HPA023881) at 1:500 overnight at 4°C. Cells were washed in PBS, incubated with an Alexa Fluor-labeled anti-rabbit secondary antibody (Thermo Fisher Scientific) at 1:500 for 2 hours at room temperature, washed in PBS, and mounted with ProLong Gold antifade reagent with DAPI (Thermo Fisher Scientific). Fluorescence images were obtained using a confocal laser-scanning microscope (A1R, Nikon). Scanning was performed in sequential laser emission mode to avoid scanning at other wavelengths.
Hpa023881
Manufactured by Merck Group
Sourced in United States
The HPA023881 is a laboratory equipment item manufactured by Merck Group. It is designed for use in scientific research and analysis. The core function of this product is to facilitate specific laboratory procedures, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence reagent, by Cytiva (1 mentions)
Ripa buffer, by Boston BioProducts (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Gradient sds page gel, by Thermo Fisher Scientific (1 mentions)
Mms 118p, by Merck Group (1 mentions)
Nb100 2220, by Merck Group (1 mentions)
Ab56416, by Novus Biologicals (1 mentions)
Sc 19992, by Abcam (1 mentions)
Lamp1, by Santa Cruz Biotechnology (1 mentions)
Lamp2, by Abcam (1 mentions)
Anti lamp 1, by BioLegend (1 mentions)
Osteopontin, by R&D Systems (1 mentions)
Filipin, by Polysciences (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
Prox1, by Merck Group (1 mentions)
Hsc70, by Santa Cruz Biotechnology (1 mentions)
Envision detection kit, by Agilent Technologies (1 mentions)
Ab19027, by Abcam (1 mentions)
Ab8978, by Abcam (1 mentions)
10 protocols using hpa023881
1
Immunofluorescence Detection of TFE3
2
Western Blot Analysis of Protein Expression
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Cells were lysed with ice-cold RIPA buffer (Boston BioProducts) in the presence of 1 × protease inhibitor cocktail (Sigma), 1 mM NaF and 1 mM Na3VO4. Total cell lysates were mixed with 2 × SDS-loading buffer and were boiled at 95 °C for 10 min. Protein samples (10–100 μg) were then loaded and separated on 4–12% gradient SDS–PAGE gels (Invitrogen) and transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h with 1% bovine serum albumin in PBS supplemented with 0.1% Tween 20 and were incubated with antibodies against GFP (1:10,000; Covance, MMS-118P), LC3 (1:2,000; Sigma, L8918), Lamin A/C (1:1,000; Cell Signaling Technology, 4777), Lamp1 (1:1,000; Developmental Studies Hybridoma Bank, H4A3), γ-tubulin (1:4,000; Sigma, T5326), TFEB (1:1,000; Cell Signaling Technology, 4240) and TFE3 (1:4,000; Sigma, HPA023881), respectively. Bound antibodies were detected using horseradish peroxidase-conjugated anti-rabbit (65-6120) or anti-mouse (62–6520) secondary antibodies (1:5,000, Invitrogen) and enhanced chemiluminescence reagent (Amersham Pharmacia Biotech). Band intensities were quantified in Image J software. Full versions of all blots are shown in Supplementary Fig. 36 .
3
Protein Extraction and Immunoblot Analysis
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Cells were lysed in a standard lysis buffer (1% Triton X-100; Cell Signaling, #9803).Where soluble and insoluble fractions are needed, samples were sonicated and centrifuged at 17,000g for 15 min and supernatant was defined as the soluble fraction. The pellet (insoluble fraction) was washed with lysis buffer, suspended in boiling SDS–PAGE loading buffer and sonicated for protein solubilization. Standard techniques were used for protein quantification, separation, transfer and blotting47 (link). The following primary antibodies were used: TFEB (Bethyl, A303-673A, 1:1,000), Cathepsin D (gift from Dr Stuart Kornfeld, Washington University, 1:4,000), Lamp1 (Santa Cruz Biotechnology, sc-19992, 1:2,000), p62/SQSTM1 (abcam, ab56416, 1:2,000), LC3 (Novus Biologicals, NB100-2220, 1:1,000), polyubiquitinated proteins (FK-2, Millipore, 04-263, 1:1,000), MiTF (Thermo Scientific, MS-771, 1:1,000) and TFE3 (Sigma Life Science, HPA023881, 1:1,000).
4
Macrophage Lysosomal Dynamics Visualized
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Primary hepatic macrophages and RAW264 macrophages were seeded on chambered cover glasses (Nalge Nunc) and stimulated as indicated. Cells were fixed with 4% paraformaldehyde and stained with anti-LAMP1 (121601; BioLegend), LAMP2 (ab13524; Abcam), Galectin-3 (14979-1-AP; Proteintech), TFE3 (HPA023881; Sigma-Aldrich), Egr1 (4153; Cell Signaling), osteopontin (AF808; R&D), and Filipin (50 μg/ml; Polysciences). Photographs were captured under identical settings for each staining. TFE3-positive ratio was quantified by counting the number of TFE3-positive nuclei out of DAPI spots.
5
Western Blot Analysis of Cultured Cells
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Western blot analysis of cultured cells or human lung samples was performed as described elsewhere33 (link). The primary antibodies used were against TFE3 (Sigma–Aldrich, HPA023881; 1:1,000), HA (Sigma–Aldrich, H6908; 1:2,000), PROX1 (Merck, 07-537; 1:1,000), SOX18 (Santa Cruz, sc-166025; 1:500), Histone H3 (Cell Signaling, 9715 S; 1:2,000), FLCN (Cell Signaling, 3697, 1/1000), and HSC70 (Santa Cruz, sc-7298; 1:500).
6
Immunohistochemical Profiling of Tumor Cells
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The 4-μm-thick sections were prepared from 10% FFPE tissue blocks for TFE3 IHC staining. All slides were exposed to 3% H2O2 for 10 minutes at room temperature to block endogenous peroxidase activity. TFE3 (HPA023881, Sigma, USA), cathepsin K (ab19027, Abcam, Cambridge, UK), CD10 (ab227640, Abcam, Cambridge, UK), CA-IX (ab107257, Abcam, Cambridge, UK), Vimentin (ab8978, Abcam, Cambridge, UK), CD117(ab32363, Abcam, Cambridge, UK), CK7 (ab181598, Abcam, Cambridge, UK) antibody were incubated with tumor sections in a humidified chamber at 4°C overnight, then the anti-mouse or anti-rabbit peroxidase-conjugated secondary antibody (EnVision™ Detection Kit, DAKO, Denmark) were used with the sections at 37°C for 30 minutes.
The result was evaluated in a semiquantitative manner by multiplying the staining intensity (0 = no staining, 1 = mild staining, 2 =moderate staining, and 3 = strong staining) by the percentage of immunoreactive tumor cells (0–100). The final immunostaining result was calculated as following: negative (0), score <25; weak positive (1+), score 26–100; moderate positive (2+), score 101–200; strong positive (3+), score 201–300.
The result was evaluated in a semiquantitative manner by multiplying the staining intensity (0 = no staining, 1 = mild staining, 2 =moderate staining, and 3 = strong staining) by the percentage of immunoreactive tumor cells (0–100). The final immunostaining result was calculated as following: negative (0), score <25; weak positive (1+), score 26–100; moderate positive (2+), score 101–200; strong positive (3+), score 201–300.
7
CAMTA1 and TFE3 IHC for Tissue
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CAMTA1 (NBP1—93620, Novusbio, Centennial, CO, USA) and TFE3 (HPA023881, Sigma-Aldrich, St. Louis, MO, USA) immunohistochemistry staining was performed on 4 µm FFPE tissue sections when both RT-PCR and FISH could not be performed or were not interpretable.
8
Immunostaining of Pluripotency Markers
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Cells were grown on Matrigel-coated glass cover slips (13 mm; Marienfeld) in 24-well plates, fixed with 4% paraformaldehyde, and incubated in blocking solution (2.5% BSA) with 0.1% Triton X-100 to enable staining of intracellular markers. Cells were incubated with primary antibody for 1 hr. at RT, washed and incubated with secondary antibodies for 1 hr. at RT, counterstained with DAPI for nuclear staining, and imaged using an Olympus IX51 inverted light microscope. The following antibodies were used at the indicated dilutions: mouse anti-OCT4 (sc-5279, Santa-Cruz, 1:60), mouse anti-SSEA4 (CST-4755S, cell signaling technology, 1:200), mouse anti-TRA-1-60 (ab16288, Abcam, 1:200) and rabbit anti-TFE3 (HPA023881, Sigma-Aldrich, 1:60).
9
TFE3 Chromatin Immunoprecipitation in tRCC
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UOK146 cells (tRCC with a PRCC-TFE3 fusion) were fixed with 1% formaldehyde and subjected to ChIP as previously described 21 using an antibody against TFE3 (Sigma Aldrich, HPA023881). ChIP-seq data were mapped to the human genome (hg19) using the Bowtie algorithm, allowing up to two mismatches. Reads mapped to more than 20 locations along the genome were discarded. ChIP-seq data generated using an IgG antibody were used as a control 22 . Peaks were identified using MACS2 with an FDR-adjusted P value cutoff of 0.05. Two biological replicates were generated 23 (link) .
10
Imaging TFE3 Nuclear Translocation in Macrophages
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To assess TFE3 nuclear translocation, wild-type RAW264.7 macrophages were left uninfected or infected with MOI = 50 of EHEC or ΔEhaF for 2 h, 3 h, or 4 h. Cells were washed 3 times with PBS, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-X, and blocked with 10% goat serum prior to incubating with anti-TFE3 antibody (1:200 dilution, HPA023881, Sigma Aldrich) overnight. The cells were then stained with fluorescently labeled secondary antibody (CF®647 conjugated Anti-rabbit, 1:200 dilution, 20282, Biotium) followed by Alexa Fluor 488 conjugated Phalloidin (1:200 dilution, A12379, Invitrogen) (for plasma membrane), and DAPI (for nucleus) and visualized with Zeiss LSM 880 microscope. Images were analyzed with ImageJ 1.53a.
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