Pa1 16601

Manufactured by Thermo Fisher Scientific

Sourced in United States

The PA1-16601 is a laboratory instrument produced by Thermo Fisher Scientific. It is designed for measuring and analyzing various samples in a controlled laboratory environment. The core function of this product is to provide precise and reliable data collection and processing capabilities for research and testing purposes.

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5 protocols using pa1 16601

Western Blot Analysis of PD-L1 and HIF-1α

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The cells were collected in Pierce^TM RIPA buffer (89901, Thermo Fisher Scientific) supplemented with 3% phosphatase and protease inhibitors (78442, Thermo Fisher Scientific) on ice and incubated for 30 min. Protein concentration was measured using the Pierce^TM BCA Protein Assay Kit (23225, Thermo Fisher Scientific) on a Nanodrop 2000 Spectrophotometer. Protein lysates (20 μg) were separated by electrophoresis on 4-20% Mini-PROTEAN^@ TGX^TM Precast Gel (#4561093, Bio-Rad) and transferred to a nitrocellulose membrane (1704271, Bio-Rad) using the Trans-Blot Turbo Transfer System (Bio-Rad).
Membranes were blocked with 5% non-fat milk solution (T145.1, ROTH) and then incubated for 1 h at RT employing either PD-L1 polyclonal antibody (PA5-20343, Invitrogen) (dilution 1:1000) or HIF-1α polyclonal antibody (PA1-16601, Invitrogen) (dilution 1:1000). β-actin (ab8227, Abcam) (dilution 1:2000) was used to standardize the loading. Polyclonal Goat Anti-Rabbit (P0448, Dako) HRP at a dilution 1:2000 was used as secondary antibody. The signals were detected utilizing Amersham ECL Western Blotting Detection Reagents (9838243, GE Healthcare, Freiburg, Germany) on Amersham Hyperfilm (28906836, GE Healthcare) on OPTIMAX X-Ray Film Processor (11701-9806-3716, PROTEC GmbH, Oberstenfeld, Germany). The ImageJ program (version 2, NIH, Washington, DC, USA) was used for densitometric analysis.

Yong J., Gröger S., von Bremen J., Meyle J, & Ruf S. (2022). Immunorthodontics: PD-L1, a Novel Immunomodulator in Cementoblasts, Is Regulated by HIF-1α under Hypoxia. Cells, 11(15), 2350.

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Histopathological Characterization of Minipig SCG Tumors

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The minipig SCG tumors and xenograft tumors were fixed overnight with 4% PFA followed by paraffin embedding. H&E staining was performed to evaluate histopathologic features in pig SCG tumors and xenografts. Immunohistochemistry was performed using primary antibodies for Ki67 (ab15580, ABCAM, USA), SOX2 (ab92494, ABCAM, USA), Olig2 (P21954, Invitrogen, USA), NG2 (ab129051, ABCAM, USA), NQO1 (#3187, Cell Signaling Technology, USA), 4-HNE (MA5-27,570, Invitrogen, USA), HIF-1α (PA1-16,601, Invitrogen, USA), GLUT-1 (ab14683, ABCAM, USA), CA-IX (66,243, Proteintech, USA) with Vectastain Kit (PK-8200, Vector Laboratories, Inc., USA). All images were acquired using whole-slide scanning at 40 × magnification (Leica Aperio AT2 Slide Scanner) [14 (link)].

Tora M.S., Neill S.G., Lakhina Y., Assed H., Zhang M., Nagarajan P.P., Federici T., Gutierrez J., Hoang K.B., Du Y., Lei K, & Boulis N.M. (2023). Tumor microenvironment in a minipig model of spinal cord glioma. Journal of Translational Medicine, 21, 667.

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Heme Proteins Modulate Macrophage Signaling

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Human macrophages were exposed to 10 μM oxyHb or 10 μM ferrylHb for 2 h. In other experiments, human macrophages were exposed to 10 μM heme, oxyHb, metHb, or ferrylHb for 16 h. The expression of PI3K (n = 3), phopsho-PI3K (n = 3), and HIF-1α (n = 5) was assessed by Western blot. After treatment, the cells were solubilized in protein lysis buffer (in case of PI3K, HIF-1α proteins) or protein lysis buffer completed with phosphatase inhibitor (in case of phopsho-PI3K). Twenty micrograms of total protein was applied to 10% SDS-PAGE gels. Gels were blotted to the PVDF membrane and blocked with 5% BSA. Proteins were incubated with the following antibodies overnight: mouse anti-human PI3K antibody (MA1-74183; dilution: 1:500; Invitrogen), rabbit anti-human phospho-PI3K antibody (PA5-104853; dilution: 1:500; Invitrogen), rabbit anti-human HIF-1α antibody (PA1-16601; dilution: 1:500; Invitrogen) followed by HRP-conjugated mouse (ab6728; Abcam Plc) or rabbit secondary antibody (ab6721; Abcam Plc) at a dilution of 1:4000 for 2 h. Human macrophages were exposed to 10 μM heme, oxyHb, metHb, or ferrylHb for 24 h. The secreted level of VEGF-A was measured with the enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (RAB0507; Sigma).

Potor L., Hendrik Z., Patsalos A., Katona É., Méhes G., Póliska S., Csősz É., Kalló G., Komáromi I., Combi Z., Posta N., Sikura K.É., Pethő D., Oros M., Vereb G., Tóth C., Gergely P., Nagy L., Balla G, & Balla J. (2021). Oxidation of Hemoglobin Drives a Proatherogenic Polarization of Macrophages in Human Atherosclerosis. Antioxidants & Redox Signaling, 35(12), 917-950.

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Co-Immunoprecipitation of HIF-1α and Slug

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Co-immunoprecipitation was performed according to the previous report [22 (link)]. A549 cells were lysed in IP lysis buffer and lysate (1 mg) was incubated for 1 h with IgG and 10 μg of protein A/G magnetic beads (Thermo Fisher Scientific Inc., Waltham, MA, USA) for preclearing. The lysate was incubated with 10 μg of anti-HIF-1α (PA1-16601, Thermo Fisher Scientific Inc., Waltham, MA, USA) and anti-Slug (SC-166476, Santacruz Biotechnology, Inc., Dallas, TX, USA) antibodies or normal mouse IgGs at 4 °C overnight. protein A/G magnetic beads were used to incubate with the lysate at RT for 1 h. The supernatants were removed carefully and the immunoprecipitates were collected in a magnetic separation rack. Pellets were washed twice with PBS and the immunoprecipitates were eluted in 40 μL of 2x SDS-PAGE reducing sample buffer and analyzed by Western blot with anti-HIF-1α or anti-Slug antibodies.

Kim H.J., Park M.K., Byun H.J., Kim M., Kim B., Yu L., Nguyen T.M., Nguyen T.H., Do P.A., Kim E.J., Kim J.H., Enkhtaivan E., Kim K.S., Jang J.Y., Kang G.J., Lee H., Won M., Lee K., Cho J, & Lee C.H. (2021). LW1497, an Inhibitor of Malate Dehydrogenase, Suppresses TGF-β1-Induced Epithelial-Mesenchymal Transition in Lung Cancer Cells by Downregulating Slug. Antioxidants, 10(11), 1674.

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Western Blotting for Protein Analysis

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For western blotting analysis, total protein from tissues was extracted using Whole Cell Lysis Assay (KGP250, Keygen biotech, Nanjing, China). Protein concentrations were measured by bicinchoninic acid (BCA) reagent (P0012 Beyotime Biotechnology) using a Molecule Devices spectrophotometer. Equal amounts of protein were resolved in NuPAGE LDS sample buffer (NP0007, Thermo Fisher Scientific) and heated at 100 °C for 5 min. After being separated by SDS–PAGE, the separated protein was transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore) which were blocked with 5% non-fat milk and incubated at 4 °C for overnight with the primary antibodies including GAPDH (ab181602, Abcam) and HIF-1α (PA1-16601, Thermo Fisher Scientific), followed by the corresponding secondary antibodies. Then the blots were detected using a ChemiScope- Touch (Clinx Science Instruments).

Wu J., Wang X., Chen L., Wang J., Zhang J., Tang J., Ji Y., Song J., Wang L., Zhao Y., Zhang H., Li T., Sheng J., Chen D., Zhang Q, & Liang T. (2022). Oxygen microcapsules improve immune checkpoint blockade by ameliorating hypoxia condition in pancreatic ductal adenocarcinoma. Bioactive Materials, 20, 259-270.

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