Cells were washed in phosphate-buffered saline (PBS) and lysed using radioimmunoprecipitation assay buffer (Invitrogen, Carlsbad, CA) supplemented with a protease inhibitor cocktail (Roche, Pleasanton, CA, USA). The protein concentration was evaluated using a bicinchoninic acid protein assay kit (Bioswamp, PAB180007, Wuhan, China). Equivalent amounts of proteins (30 μg) from each sample were subjected to sodium dodecyl-polyacrylamide electrophoresis, transferred to a polyvinylidene fluoride membrane, blocked in 5% fat-free milk for 2 hours at room temperature, and incubated with the following primary antibodies: PTEN (ab32199, 1:10,000, abcam), PI3K (ab191606, 1:10,000, abcam), p-PI3K (ab182651, 1:10,000, abcam), mTOR (ab32028, 1:10,000, abcam), p-mTOR (ab109268, 1:10,000, abcam), GLUT1 (ab652, 1:10,000, abcam), LDHB (ab85319, 1:10,000, abcam), HK2 (ab209847, 1:10,000, abcam), PKM2 (ab150377, 1:10,000, abcam), or GAPDH (PAB36264, 1:10,000, Bioswamp). Then, the membranes were washed with Tris-buffered saline and incubated with goat anti-rabbit IgG secondary antibody (SAB43711, 1:10,000, Bioswamp) for 2 h at room temperature. An enhanced chemiluminescence kit (Pierce) was used to detect specific bands and autoradiograms were quantified by densitometry (Quantity One software, Bio-Rad, Hercules, CA, USA) using GAPDH as a control. For each group, the quantification was triplicated.
Ab85319
Manufactured by Abcam
Sourced in United Kingdom
Ab85319 is a laboratory equipment product manufactured by Abcam. It serves as a core function for specific applications in research and scientific experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Ab47010, by Abcam (2 mentions)
Radioimmunoprecipitation assay buffer, by Thermo Fisher Scientific (1 mentions)
Ab32028, by Abcam (1 mentions)
Ab109268, by Abcam (1 mentions)
Ab32199, by Abcam (1 mentions)
Ab652, by Abcam (1 mentions)
Ab209847, by Abcam (1 mentions)
Ab150377, by Abcam (1 mentions)
Ab182651, by Abcam (1 mentions)
Ab191606, by Abcam (1 mentions)
Pab36264, by Bioswamp (1 mentions)
Sab43711, by Bioswamp (1 mentions)
Pab180007, by Bioswamp (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ab93048, by Abcam (1 mentions)
Ab150423, by Abcam (1 mentions)
Proteinase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Anti rabbit hrp secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ab101562, by Abcam (1 mentions)
6 protocols using ab85319
1
Western Blot Analysis of Signaling Proteins
2
Protein Expression Analysis of Rabbit Disc Tissues
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AF and NP tissues were carefully isolated separately from lumbar discs of four 6-month-old female New Zealand White rabbits. Tissue protein extracts were prepared using T-PER Tissue Protein Extraction Reagent with proteinase inhibitor cocktail as per the manufacturers instructions (Cat. No 78510, Thermo Fisher). Western blots were performed as described previously [22 (link)] to detect hexokinase-1 (Anti-HK, Ab150423, Abcam), MCT4 (anti-MCT4, SC-376140, Santa Cruz Biotechnolgy), LDHA (anti-LDHA, PA5-27406, Invitrogen), MCT1 (anti-MCT1, AB93048, Abcam), LDHB (anti-LDHB, Ab85319, Abcam), and pyruvate dehydrogenase (PDH; anti-PDH, #2784S, Cell Signaling Technology). Loading control -actin (Cat. No. PA1-183, Thermo Fisher) and anti-rabbit HRP secondary antibody (Cat. No. 31460, Thermo Fisher) were used.
3
Protein Expression Analysis via Western Blot
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Tissues and whole-cell lysates were extracted for total protein using SDS buffer. Protein concentrations were determined using a BCA protein assay kit (KeyGEN BioTECH Laboratories, China) according to the manufacturer’s protocol. Equal amounts of protein were subjected to western blotting analysis. Blots were probed with antibodies against FUBP1 (ABE1330, Merck Millipore, USA), LDHB (ab85319, Abcam, USA), LDHA (ab101562, Abcam, USA), VHL (ab140989, Abcam, USA), HIF1α (14,179 s, CST, USA), HK2 (#2106 s, CST, USA), c-Myc (13,987 s, CST, USA), N-Myc (84,406, CST, USA) and β-actin (A5441, Sigma, USA). HRP-conjugated anti-rabbit (Santa-Cruz, USA) and anti-mouse (Santa-Cruz, USA) secondary antibodies were used. Proteins were determined using ECL Plus Reagent (Merck Millipore, USA).
4
Western Blot Analysis of LDH Isoforms
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Western blot analysis was done following a previously described procedure [43 (link)]. Antibodies against lactate dehydrogenase monomers and beta-actin were purchased from Abcam (Cambridge, United Kingdom) and used in the following concentrations: LDHA (1 µg/mL; ab47010), LDHB (0.1 µg/mL; ab85319), and β-actin (0.5 µg/mL; ab8226). Prior to loading onto the gel, nine samples from each group were pooled by three to obtain three final samples per group (Figure 2 shows two representative bands for each group cut from the same blot, for full blots, see Supplementary Information Figure S2 ). ImageJ software (National Institute of Health, Bethesda, Maryland, United States) was used for densitometric analyses of immunoreactive bands. Original band density was calculated as the sum of pixel intensities within a band. Final band density was obtained as the ratio of dots per band for the target protein averaged against β-actin (gel loading control). Protein expression is expressed in arbitrary units (AU) from three independent experiments.
5
Protein Expression Analysis in Prostate Cancer Cells
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Total proteins were extracted from PC3 and 22RV1 cells with RIPA buffer (Beyotime, China). Protein samples were separated via 12% SDS‒PAGE and subsequently transferred to PVDF membranes (Millipore, USA). The PVDF membranes were blocked with 5% nonfat milk and then probed with the following primary antibodies: anti-LDHA (#2012, 1:1000; Cell Signaling Technology, Danvers, MA); anti-LDHB (ab85319, 1:1000; Abcam, Cambridge, UK); anti-STAT1 (ab234400, 1:1000; Abcam); anti-FGF1 (ab207321, 1:1000; Abcam); anti-FGF2 (ab208687, 1:1000; Abcam); and anti-GAPDH (ab9485, 1:2500; Abcam) overnight at 4 °C. Afterwards, the sections were incubated with secondary antibodies (Abcam, ab7090, 1:2000). The protein bands were detected with an enhanced chemiluminescence (ECL) kit (Millipore) and quantified using ImageJ software.
6
Immunolabeling of LDHA and LDHB
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Standard immunolabeling procedure was conducted as previously described [44 (link)] using primary antibodies against LDHA (1 µg/mL; ab47010) and LDHB (0.1 µg/mL; ab85319) and the appropriate mix of secondary antibodies (all purchased from Abcam, Cambridge, UK). Briefly, antigen retrieval was performed in citrate buffer on 4% PFA fixed, paraffin-embedded, 5µm serial sections. Incubation with primary antibodies was performed overnight at 4 °C, followed by incubation with secondary antibodies for 2 h at room temperature. Immunodetection was performed using the Dako LSAB Universal Kit (Agilent Technologies, Santa Clara, CA, USA). The sections were counterstained with hematoxylin, mounted, and examined with a Leica DMLB microscope (Leica Microsystems, Wetzlar, Germany).
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