Human/Mouse/Rat HTRA2/Omi Antibody

Detection of Human/Mouse/Rat HTRA2/Omi by Western Blot. Western blot shows lysates of PC-12 rat adrenal pheochromocytoma cell line, Jurkat human acute T cell leukemia cell line, HeLa human cervical epithelial carcinoma cell line, L-929 mouse fibroblast cell line, and C2C12 mouse myoblast cell line. PVDF membrane was probed with 0.25 µg/mL of Rabbit Anti-Human/Mouse/Rat HTRA2/Omi Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1458) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). Specific bands were detected for HTRA2/Omi at approximately 36 and 49 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 2.

HTRA2/Omi in Jurkat Human Cell Line. HTRA2/Omi was detected in immersion fixed Jurkat human acute T cell leukemia cell line stimulated with staurosporin using Rabbit Anti-Human/Mouse/Rat HTRA2/Omi Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1458) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody (yellow; Catalog # NL004) and counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Detection of Human and Mouse HTRA2/Omi by Simple Western^TM. Simple Western lane view shows lysates of C2C12 mouse myoblast cell line and HeLa human cervical epithelial carcinoma cell line, loaded at 0.2 mg/mL. Specific bands were detected for HTRA2/Omi at approximately 51 kDa (precursor) and 41 kDa (processed) (as indicated) using 2.5 µg/mL of Rabbit Anti-Human/Mouse/Rat HTRA2/Omi Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1458). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Detection of Human HTRA2/Omi by Western Blot Caspases are not activated by FGFR inhibition in FGFR2‐mutant EC cells. (A) Western blots showing total caspase‐3 and caspase‐7 in response to treatment with 300 nm BGJ398 for up to 72 h. Tubulin serves as a loading control. *Denotes nonspecific band. (B) AN3CA and JHUEM2 cells were pretreated with 100 μm Z‐VAD‐FMK for 1 h prior to the addition of DMSO, 1 μm PD173074 (PD), 300 nm BGJ398 (BGJ) or 300 nm AZD4547 (AZD) for 72 h. Cell death was detected by staining cells with Annexin V. The mean percentage of Annexin V‐positive cells from three independent experiments (each performed in triplicate) is shown along with SD. (C) Western blot showing cleavage of caspase‐3 in AN3CA and JHUEM2 cells treated with 1 μm actinomycin D (Act D) or staurosporine (STS), respectively, for 24 h. (D) Mean percentage of AN3CA and JHUEM2 cells showing Annexin V‐positive staining following treatment with 100 μm Z‐VAD‐FMK alone or 1 h prior to treatment with 1 μm Act D and STS for 24 h. The mean from three independent experiments (each performed in triplicate) is shown along with SD. (E) Western blot showing staining of Bim, Bid, HTRA2/OMI and Smac/diablo in cytosolic and mitochondrial fractions of JHUEM2 cells treated with 300 nm BGJ398 for 24 and 48 h. Tom20 serves as a marker of the mitochondrial fraction, GRP78 as a marker of the ER, Lamin B1 as a marker of the nuclear fraction and GAPDH as a marker of the cytosolic fraction. (F) Western blot showing staining of AIF in mitochondrial and nuclear fractions of AN3CA cells treated with 1 μm PD173074 for 48 h. Cox IV and PARP serve as mitochondrial and nuclear markers, respectively. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/30537101), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse HTRA2/Omi by Western Blot Neural deletion of Htra2 is sufficient to generate neurological phenotypes.(A) Exons 2 to 4 of Htra2 were flanked with loxP sites, with a FRT flanked neo cassette 3′ to exon 4. Expression of FlpE causes deletion of the selection cassette. Cre-mediated deletion causes excision of exons 2 to 4. Small arrows beneath the allele constructs denote the position of genotyping primers. (B) PCR from genomic DNA can distinguish WT (+, arrow, 279 bp), KO (–, filled arrowhead, 358 bp) and floxed (f, empty arrowhead, 313 bp) alleles of Htra2. (C) Western blot analysis confirmed loss of HTRA2 protein (arrow) in all tissues of HTRA2 KO mice and reduction in brain of NesKO mice (arrowheads denote non-specific bands). The levels of HTRA2 protein in NesKO spleen and thymus were comparable with NesWT. Cx: cortex, Mb: midbrain, Hb: hindbrain. PHB2 was used as a loading control. (D) HTRA2 KO mice and NesKO mice were smaller than WT littermates by comparison. The size of the thymus and spleen was reduced although brain was relatively normal in size (representative animals shown at P30, scale bar: 1 cm.). (E) Body weight of HTRA2 KO and NesKO mice did not increase beyond P18 (n = 56 (HTRA2 WT), 62 (HTRA2 KO), 35 (NesWT), 25 (NesKO), error bars indicate SEM). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/25531304), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse HTRA2/Omi by Western Blot Neural deletion of Htra2 is sufficient to generate neurological phenotypes.(A) Exons 2 to 4 of Htra2 were flanked with loxP sites, with a FRT flanked neo cassette 3′ to exon 4. Expression of FlpE causes deletion of the selection cassette. Cre-mediated deletion causes excision of exons 2 to 4. Small arrows beneath the allele constructs denote the position of genotyping primers. (B) PCR from genomic DNA can distinguish WT (+, arrow, 279 bp), KO (–, filled arrowhead, 358 bp) and floxed (f, empty arrowhead, 313 bp) alleles of Htra2. (C) Western blot analysis confirmed loss of HTRA2 protein (arrow) in all tissues of HTRA2 KO mice and reduction in brain of NesKO mice (arrowheads denote non-specific bands). The levels of HTRA2 protein in NesKO spleen and thymus were comparable with NesWT. Cx: cortex, Mb: midbrain, Hb: hindbrain. PHB2 was used as a loading control. (D) HTRA2 KO mice and NesKO mice were smaller than WT littermates by comparison. The size of the thymus and spleen was reduced although brain was relatively normal in size (representative animals shown at P30, scale bar: 1 cm.). (E) Body weight of HTRA2 KO and NesKO mice did not increase beyond P18 (n = 56 (HTRA2 WT), 62 (HTRA2 KO), 35 (NesWT), 25 (NesKO), error bars indicate SEM). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/25531304), licensed under a CC-BY license. Not internally tested by R&D Systems.