- PA5-17325 - Invitrogen Antibodies PHB antibody

Alula KM, Delgado-Deida Y, Jackson DN, Venuprasad K, Theiss AL
Oncogene 2021 Jan;40(2):369-383

Al Rahim M, Thatipamula S, Pasinetti GM, Hossain MA
ASN neuro 2021 Jan-Dec;13:17590914211012888

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of PHB1 in extracts from PC12 and COS cells using PHB1 polyclonal antibody (Product # PA5-17325).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of Prohibitin was achieved by transfecting A-431 with Prohibitin specific siRNAs (Silencer® select Product # s10425, s10424). Western blot analysis (Fig. a) was performed using Whole cell extracts from the Prohibitin knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with Prohibitin Polyclonal Antibody (Product # PA5-17325, 1:1000 dilution) 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 Prohibitin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Prohibitin Polyclonal Antibody(Product # PA5-17325) and a 30 kDa band corresponding to Prohibitin was observed across cell lines and tissues tested. Whole cell extracts (30 µg lysate) of Caco-2 (Lane 1), Hep G2 (Lane 2), T-47D (Lane 3), A-431 (Lane 4), HeLa (Lane 5), NIH/3T3 (Lane 6), PC-12 (Lane 7) and tissue extracts (30 µg) of Mouse Heart (Lane 8), Rat Brain (Lane 9) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0341BOX). 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 (1:1000 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 Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Prohibitin was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 45 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Prohibitin Polyclonal Antibody (Product # PA5-17325) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing cytoplasm and nucleus localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1. PHB1 expression inversely correlates with beta-catenin expression in adenomas. Western blots of nuclear and cytosolic protein fractions of isolated IECs from jejunum (A) or mucosa of colon (B) of wild-type (WT) and Apc Min/+ mice or isolated adenomas from Apc Min/+ mice. *denotes non-specific band in (A), likely family member PHB2. Mean +- SEM of PHB1 western blot densitometry in jejunum (C) or colon (D). Mean +- SEM of beta-catenin western blot densitometry in jejunum (E) or colon (F). (G) Spearman rank correlation of western densitometry in nuclear fraction of adenomas. n = 3 mice per group. * P < 0.05, ** P < 0.01 vs. WT IECs by one-way ANOVA followed by Bonferroni's test.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 8. Relative expression of PHB1 regulates beta-catenin activation and cell viability in RKO and SW48 CRC cell lines. (A-C) RKO or SW48 cells were transfected with pCDNA4 (vector; V), full-length pCDNA4-PHB1 (FL), or pCDNA4-PHB1 DeltaNES (DeltaNES) for 72 hr. (A) Immunofluorescent staining of RKO cells. Scale bars: 20 mum; boxed pullouts: 20 mum. (B) Mean +- SEM of relative AXIN1 mRNA expression. (C) Cell viability as measured by LDH release. (D-G) RKO or SW48 cells were transfected with 2 unique siRNAs against PHB1 or siNegative Control (siNC) for 48 hr. (D) Mean +- SEM of relative AXIN1 mRNA expression. (E) Representative western blot of beta-catenin, AXIN1, or PHB1 (to demonstrate efficiency of siRNA knockdown). (F) Mean +- SEM of densitometric units by western blotting. (G) Relative luciferase expression of transcriptional activation by beta-Catenin after 10 muM XAV939 or vehicle treatment for 16 hr. Negative control (NC) cells were transfected with a non-inducible firefly reporter construct. n = 3 (A, D-G) or n = 6 (B, C) biological replicates per group. * P < 0.05, ** P < 0.01 vs V by unpaired, two-tailed Student's t test (B, C); * P < 0.05 vs siNC vehicle, # P < 0.05, ## P < 0.01 vs siPhb1 vehicle by one-way ANOVA (D, F) or two-way ANOVA followed by Bonferroni's test (G).

PA5-17325