- PA5-30575 - Invitrogen Antibodies POLR1D antibody

Gennaro VJ, Wedegaertner H, McMahon SB
BMC cancer 2019 Mar 22;19(1):258

No comments: Submit comment

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on modified whole cell extracts (1% SDS) (30 µg lysate) of HeLa (Lane 1), MCF7 (Lane 2), A549 (Lane 3), U-87MG (Lane 4), NTERA-2 (Lane 5) and HepG2 (Lane 6). The blot was probed with Anti-POLR1D Polyclonal Antibody (Product # PA5-30575, 1:3000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 20 kDa band corresponding to POLR1D was observed in all cell lines tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot using POLR1D Polyclonal Antibody (Product # PA5-30575). Sample (30 µg of whole cell lysate). Lane A: Hep G2 . 15% SDS PAGE. POLR1D Polyclonal Antibody (Product # PA5-30575) diluted at 1:1,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of POLR1D was achieved by transfecting HeLa cells with POLR1D specific siRNAs (Silencer® select Product # s234268). Western blot analysis (Fig. a) was performed using whole cell extracts from the POLR1D knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with POLR1D Polyclonal Antibody (Product # PA5-30575, 1:3000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 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 POLR1D.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of POLR1D was performed using 70% confluent log phase A549 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with POLR1D Polyclonal Antibody (Product # PA5-30575) at 1:200 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415). Panel d represents the merged image showing Nuclear 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)
Main image
Experimental details: Immunohistochemical analysis of paraffin-embedded HBL435 xenograft, using POLR1D (Product # PA5-30575) antibody at 1:500 dilution. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Chromatin immunoprecipitation analysis of POLR1D was performed using cross-linked chromatin from 1x10^6 HCT116 colon carcinoma cells treated with serum for 0 and 30 minutes. Immunoprecipitation was performed using a multiplex microplate Matrix ChIP assay (see reference for Matrix ChIP protocol: http://www.ncbi.nlm.nih.gov/pubmed/22098709) with 1.0 µL/100 µL well volume of a POLR1D polyclonal antibody (Product # PA5-30575). Chromatin aliquots from ~1x10^5 cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1 µL of eluted DNA in 2 µL SYBR real-time PCR reactions containing primers to amplify exon-1 of the RNA5-8S5 gene or -20 kb upstream of the LAMC1 gene. PCR calibration curves were generated for each primer pair from a dilution series of sheared total genomic DNA. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. Results represent the mean +/- SEM for three experiments. A schematic representations of the RNA5-8S5 and LAMC1 loci are shown above the data where boxes represent exons (black boxes = translated regions, white boxes = untranslated regions), the zigzag line represents an intron, and the straight line represents upstream sequence. Regions amplified by RNA5-8S5 and LAMC1 primers are represented by black bars. Data courtesy of the Innovators Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 4 Proteomic analysis identified proteins upregulated in the presence of pro-survival HSP70/BAG1S complex. a U2OS MYC-ER cells expressing ectopic vector, BAG1S, or BAG1DeltaS depleted of endogenous BAG1 protein. MYC activity induced for 12 or 24 h with +-100 nM 4-OHT treatment. Lysates analyzed via IB to detect changes in known HSP70 chaperone client proteins GCR, XIAP and RAF1. b Schematic of experimental conditions representing endogenous BAG1 (vector - shLUC), BAG1 knockdown (vector - shBAG1), BAG1S only (BAG1S - shBAG1), or BAG1DeltaS only (BAG1DeltaS - shBAG1) evaluated for differences in global protein levels. Proteomics analysis outlined with exclusion criteria for significant protein differences between samples. c Efficient knockdown of endogenous BAG1 and rescue of BAG1S and BAG1DeltaS shown by IB for samples subjected to proteomics analysis. d Proteomic hits assessed based on schematic of compiled proteins with >=|1.5| fold change in knockdown compared to control and p =10% constitutes a partial rescue. Overlapping proteins with BAG1S or BAG1DeltaS indicative of proteins rescued by either ectopic protei

PA5-30575