- PA5-34360 - Invitrogen Antibodies TFEB antibody

Submitted references Transcriptional regulation of autophagy-lysosomal function in BRAF-driven melanoma progression and chemoresistance.

Li S, Song Y, Quach C, Guo H, Jang GB, Maazi H, Zhao S, Sands NA, Liu Q, In GK, Peng D, Yuan W, Machida K, Yu M, Akbari O, Hagiya A, Yang Y, Punj V, Tang L, Liang C
Nature communications 2019 Apr 12;10(1):1693

HSPA5 negatively regulates lysosomal activity through ubiquitination of MUL1 in head and neck cancer.

Kim SY, Kim HJ, Kim HJ, Kim DH, Han JH, Byeon HK, Lee K, Kim CH
Autophagy 2018;14(3):385-403

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of A549 cell lysate using a TFEB polyclonal antibody (Product # PA5-34360) at (A) 1 and (B) 2 µg/mL.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot analysis of TFEB in A549 cell lysate with TFEB Polyclonal Antibody (Product # PA5-34360) at (A) 1 and (B) 2 µg/mL.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of TFEB was achieved by transfecting Raji with TFEB specific siRNAs (Silencer® select Product # s15495, s15496). Western blot analysis (Fig. a) was performed using whole cell extracts from the TFEB knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with TFEB Polyclonal Antibody (Product # PA5-34360, 1µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to TFEB.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-TFEB Polyclonal Antibody (Product # PA5-34360) and a 55kDa band corresponding to TFEB was observed across all cell lines tested except Jurkat, MOLT-4 and U-937 which are known to have low TFEB expression (DOI: 10.1128/mcb.10.8.4384). Whole cell extracts (30 µg lysate) of Raji (Lane 1), Daudi (Lane 2), Ramos (Lane 3), Jurkat (Lane 4), MOLT-4 (Lane 5) and U-937 (Lane 6) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1 µg/mL dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of human lung tissue using a TFEB polyclonal antibody (Product # PA5-34360) at a 20 µg/mL dilution.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence of TFEB in human lung tissue with TFEB Polyclonal Antibody (Product # PA5-34360) at 20 µg/mL.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry of TFEB (NT) in human lung tissue with TFEB Polyclonal Antibody (Product # PA5-34360) at 2.5 µg/mL.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 1 BRAF V600E inhibitor triggers autophagy-lysosomal activation through TFEB. a , b Western blot analysis ( a ) and densitometric quantification ( b ) of the LC3-II/LC3-I and the p62/Actin ratios in A375 and MeWo cells treated with the indicated concentrations of PLX4720 for 12 h. Actin served as a loading control. n = 4 independent experiments. c Representative images of LysoTracker Red staining of A375 and MeWo cells treated for 12 h with DMSO or PLX4720 (1 muM). Quantification of relative fold induction of lysosomes by PLX4720 is shown in the right panel. n = 3 independent experiments. d Relative lysosome NAG activity in PLX4720-treated A375 cells. n = 3 independent experiments. e Expression analysis of the autophagy-lysosome relevant genes in PLX4720-treated A375 cells in the presence or absence of TFEB (shRNA). n = 3 independent experiments. f , g Western blot analysis ( f ) and densitometric quantification ( g ) of the LC3-II/LC3-I and p62/Actin ratios in PLX4720-treated A375 cells with shRNA-mediated depletion of the indicated genes. Expression of indicated proteins is also shown. Actin served as a loading control. n = 3 independent experiments. h , i Representative images ( h ) and quantification ( i ) of LysoTracker Red (red) and LAMP1 (green) immunostaining of PLX4720 (1 muM, 12 h-treated) A375 cells with depletion of the indicated genes. Note the reduced lysosome staining in PLX4720-treated cells upon TFEB depletion. n =

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 5 Effect of TFEB S142 phosphorylation on BRAF V600E melanoma progression. a NOD/SCID mice bearing A375 xenografts tumors stably expressing vector, WT, TFEB S142A or TFEB S142E isolated on day 34 post-injection. b , c Tumor volume ( b ) and Tumor weight (g) upon autopsy at day 34 of A375 xenografts stably expressing the indicated TFEB. n = 5-6 mice per group per time point. d H&E and immunohistochemical (IHC) staining of Ki67, active Caspase 3, 4-HNE, and 8-oxo-dG in indicated A375 tumor genotypes. The levels of mitotic figures, necrosis, and multinucleated cells were quantified (right panels; n = 5-6 mice per group). N necrotic area, T tumor. Scale bars, 100 mum. e EM images of the indicated xenograft tumor genotypes. M mitochondria. Asterisks denote damaged mitochondria. Arrows denote autophagic vacuoles. Damaged mitochondria is quantified (bottom; n = 50). Scale bar, 200 nm. f IB of EMT-related factors and TGF-beta/TGF-betaR2/Smad2/3 signaling in indicated A375 xenograft tumor genotypes (two randomly chosen samples per group). Also shown (bottom) is the relative expression of indicated proteins in xenograft tumors. n = 3 independent experiments. g GSEA plot of A375 tumors showing the significantly changed autophagy-lysosome genes. The relevant complete GSEA data are in Supplementary Data 1a . FDR false discovery rate, NES normalized enrichment score. h Heat map depicting the expression of TGF-beta target genes in indicated xenograft tum

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 8 TFEB S142 phosphorylation confers BRAFi resistance in melanoma cells. a Representative images (left) and quantification (right) of the colonogenic survival of A375 cells stably expressing control shRNA (pGIPZ) or TFEB shRNA complemented with empty vector, WT TFEB, and S142A or S142E TFEB mutants that are shRNA-resistant, after treatment with DMSO or PLX4720 at the indicated concentrations. Endogenous and reconstituted TFEB expression was confirmed by immunoblotting (bottom right). n = 3 independent experiments. b Bioluminescence images (top) of tumor regression of the indicated A375 R xenograft tumor genotype in live NOD/SCID mice at the indicated time after inoculation. Radiant efficiency expressed as p/s/cm 2 /sr/(muW/cm 2 ) was quantified (bottom). c Effect of PLX4720 on tumor response of xenografts formed by the indicated A375 cell lines. After tumor establishment (~ 500 mm 3 ), mice bearing A375 xenografts were treated with PLX4720 (20 mg/kg, i.p.) daily for 21 days. Values are the mean tumor volume +- SD per time point for 6-7 mice per group. d , e Representative gross images ( d ) of livers with metastatic nodules (top), H&E-stained sections (second row) and IHC analysis of TGF-beta and p-Smad3 of the indicated A375 xenograft genotypes 21 days after PLX4720 treatment (20 mg/kg, i.p., daily). Scale bars, 100 mum. The numbers of metastatic nodules in the liver in ( d ) was quantified in ( e ) ( n = 5-6 mice per group). For all quantification, data

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