PD-1 downstream signaling molecules in CD4+ and CD8+ T cells were measured by Western blot analysis at 96 hpi. The primary antibodies were PI3K (ab227204, 1:1000, Abcam), p-PI3K (ab182651, 1:1000, Abcam), AKT (#9272, 1:1000, Cell Signaling Technology, USA), p-AKT (Ser473) (#9271, 1:1000, Cell Signaling Technology), caspase 9 (ab202068, 1:1000, Abcam), caspase 3 (19677-1-AP, 1:500, Proteintech), ERK (#4695S, 1:1000, Cell Signaling Technology), p-ERK (Thr202/Tyr204) (#9101, 1:1000, Cell Signaling Technology), mTOR (ab2732, 1:1000, Abcam), p-mTOR (ab84400, 1:1000, Abcam), and β-actin (60008-1-lg, 1:15000, Proteintech), which was used as an internal control. The secondary antibodies were HRP-conjugated affinipure goat anti-mouse IgG (H+L) (SA00001-1, 1:8000, Proteintech) and HRP-conjugated affinipure goat anti-rabbit IgG (H+L) (SA00001-2, 1:8000, Proteintech).
Ab227204
Manufactured by Abcam
Sourced in United States
Ab227204 is a laboratory equipment product. It serves a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab182651, by Abcam (6 mentions)
Ab2732, by Abcam (4 mentions)
Sa00001 1, by Proteintech (3 mentions)
Ab38449, by Abcam (3 mentions)
Ab84400, by Abcam (2 mentions)
P erk thr202 tyr204, by Cell Signaling Technology (2 mentions)
P akt ser473, by Cell Signaling Technology (2 mentions)
P pi3k, by Abcam (2 mentions)
Sa00001 2, by Proteintech (2 mentions)
Ab59348, by Abcam (2 mentions)
Ab202068, by Abcam (1 mentions)
Hrp conjugated affinipure goat anti rabbit igg h l, by Proteintech (1 mentions)
Hrp conjugated affinipure goat anti mouse igg h l, by Proteintech (1 mentions)
Ab214424, by Abcam (1 mentions)
Ab53519, by Abcam (1 mentions)
Ab27568, by Abcam (1 mentions)
Ab76011, by Abcam (1 mentions)
Ab219332, by Abcam (1 mentions)
Ab51032, by Abcam (1 mentions)
Ab8277, by Abcam (1 mentions)
10 protocols using ab227204
1
PD-1 Signaling Pathway in T Cells
2
Western Blot Analysis of EMT and Angiogenesis Markers
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Hep3b cells were washed in PBS and lysed using the protein extraction reagent RIPA (Invitrogen; Thermo Fisher Scientific, Inc.). The concentration of proteins was measured by BCA kit (cat. no. ab102536; Abcam). Equivalent amounts of proteins (30 µg) from each sample were electrophoresed on SDS-polyacrylamide gel (SDS-PAGE) and transferred onto a polyvinylidene fluoride membrane, blocked in 4% skim milk for 2 h at room temperature and incubated with the following specific primary antibodies: E-cadherin antibody (ab219332 1:1,000; Abcam), α-catenin antibody (ab51032 1:2,000; Abcam), N-cadherin (ab76011, 1:5000 dilution, Abcam), vimentin (ab92547 1:1,000; Abcam), p-PI3K (ab182651 1:1,000; Abcam), PI3K (ab227204 1:1,000; Abcam), p-AKT (ab38449, 1:500 dilution, Abcam), AKT (ab18785 1:1,000; Abcam), VEGF (ab214424 1:1,000; Abcam), VEGFR2 (ab221679 1:1,000; Abcam), Snail (ab53519 1:1,000; Abcam), Slug (ab27568 1:1,000; Abcam) and MPP9 (ab38898 1:1,000; Abcam) overnight at 4°C. β-actin (ab8277 1:1,000; Abcam) was used as internal reference. Then, the membranes were incubated in HRP-linked goat anti-rabbit IgG secondary antibody (ab97051; 1:10,000; Abcam) for 2 h at room temperature. Immunoreactivity was visualized by a colorimetric reaction using an ECL substrate buffer (EMD Millipore) and membranes were scanned with Gel Doz EZ imager (Bio-Rad Laboratories, Inc.).
3
Molecular Analysis of Signaling Pathways
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qPCR and Western blot were performed as previously reported 19 (link). qPCR was carried out using the Hairpin-itTM microRNA qPCR Quantitation Kit (GenePharma, Shanghai, China) and LightCycler 96 (Roche, Germany). U6 snRNA was used as an internal control for miR-92b, and the mRNA levels of PTEN, TNF-α and IL-6 were normalized to GAPDH. The 2-ΔΔCt method was employed to calculate relative expression of each gene. For Western blot analysis, the protein blots were incubated with following antibodies: rabbit anti-TLR4 (1:1000, 7074, Cell Signaling Technology, USA), rabbit anti-phospho-NF-κB p65 (1:1000, 3033, Cell Signaling Technology, USA), rabbit anti-NF-κB p65 (1:1000, 4764, Cell Signaling Technology, USA), rabbit anti-β-actin (1:1000, 4970, Cell Signaling Technology, USA), rabbit anti-Bax (1:200, bs-0127R, Bioss, China), rabbit anti-Bcl-2 (1:200, bs-4563R, Bioss, China), rabbit anti-phospho-PI3K (1:200, bs-3332R, Bioss, China), rabbit anti-PI3K (1:1000, ab227204, Abcam, USA), rabbit anti-phospho-AKT (1:1000, ab8932, Abcam, USA), rabbit anti-AKT (1:500, GB111114, Servicebio, China), rabbit anti-β-catenin (1:5000, ab196204, Abcam, USA). The optical density (OD) value of each protein band was quantified using the Image-Pro Plus 6.0 (IPP 6.0) software (Media Cybernetics Inc., USA) and normalized to β-actin.
4
Protein Expression Analysis via Western Blot
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Western blot analysis to assess protein expression was performed as previously described [23 (link)]. Frozen hearts were mixed in RIPA buffer (with protease inhibitor), centrifuged (10,000 rpm, 8 min, 4 °C), and then the supernatant was assessed using a NanoDropLite spectrophotometer (Thermo Fisher Scientific, Austin, TX, USA) to determine the amount of protein. Protein samples (45 µg) were electrophoresed in SDS-PAGE, transported to the polyvinylidene fluoride (PDVF) membrane, incubated with 5% BSA and then with primary antibodies of Keap1 (ab139729), Nrf2 (ab92946), HO-1 (ab189491), PI3K (ab227204), p-PI3K (ab182651), Akt (ab210454), p-Akt (ab38449), mTOR (ab134903), p-mTOR (ab137133) and housekeeping anti-β actin (ab8227), which were obtained from Abcam (Boston, MA, USA) and diluted up to 1:1000. These primary antibodies were identified using horseradish peroxidase (HRP)-conjugated secondary antibodies. Antigen-antibody reactions were visualized with an enhanced chemiluminescence kit (ECL, Sigma-Aldrich) under a gel documentation system. Image J software (National Institutes of Health, University of Wisconsin, Madison, WI, USA) was used to evaluate the acquired images.
5
Protein Expression Analysis by Western Blot
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RIPA was applied to extract protein from cells. BCA was applied to assess the protein concentration. Subsequently, SDS-PAGE gel (10%) was applied to separate the proteins, and then proteins were transferred onto PVDF membranes. Primary antibodies were applied to incubate the membranes overnight after blocking for 1 h. Afterward, a secondary anti-rabbit antibody (SA00001-1; Proteintech, USA, 1:5000) was applied to incubate the membranes for 1 h. The primary antibodies were as follows: anti-Bax (ab32503, Abcam, Cambridge, MA, USA; 1:1000), anti-Caspase-3 (ab32351, Abcam; 1:5000), anti-Bcl-2 (ab59348, Abcam; 1:1000), anti-p-PI3K (ab182651, Abcam; 1:1000), anti-PI3K (ab227204, Abcam; 1:1000), anti-p-AKT (ab81283, Abcam; 1:5000), anti-AKT (ab108266, Abcam; 1:1000), anti-p-mTOR (ab109268, Abcam; 1:1000), anti-FOS (ab208942, Abcam; 1:1000), anti-JUN (66313-1-Ig, Proteintech; 1:1000), anti-TGFB1 (21898-1-AP, Proteintech; 1:1000), anti-PPARD (ab178866, Abcam; 1:1000), anti-mTOR (ab2732, Abcam; 1:2000) and anti-β-actin (66009-1-Ig, Proteintech; 1:5000). β-actin was applied for normalization.
6
Protein Quantification and Immunoblotting Protocol
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The cell lysis was performed using a protease suppressor cocktail (Roche) in a RIPA buffer (pH 8.0) containing NP-40 (1%), SDS (0.1%), Tris-HCl (50 mM), and NaCl (150 mM). A BCA Protein Quantitation Kit was employed for the quantification of proteins. These protein samples were isolated on SDS-PAGE gels (10%), and then, the protein bands were transferred onto PVDF membranes (0.45 μm). The membranes were then blocked for 60 min using PBST containing 5% BSA at an ambient temperature. Afterwards, these membranes were incubated for 60 min with specific primary antibodies at 4°C. The primary antibodies used were listed here: Bcl-2 ab (1:1000, ab59348, Abcam), Bax ab (1:1000, ab227754, Abcam), LC3B-I ab (1:500, ab48394, Abcam), p62 ab (1:2000, ab56416, Abcam), PI3K ab (1:2500, ab227204, Abcam), mTOR ab (1:1000, ab2732, Abcam), Akt ab (1:2000, ab8805, Abcam), pAkt ab(1:500, ab38449, Abcam), and GAPDH (1:5000, ab8245, Abcam) Then, the membranes were incubated with the secondary antibodies conjugated with Amersham ECL peroxidase: goat anti-rabbit IgG (1:5,000) or goat anti-mouse IgG (1:5,000) at an ambient temperature for another 60 min. The immunoreactivity was measured using a Super Signal West Femto Maximum Sensitivity Substrate Kit (provided by Thermo) on a C-DiGit Blot Scanner.
7
Western Blot Analysis of Osteogenic Markers
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After 7 and 14 days of co-culture with the material (n = 5), the cells were lysed with RIPA lysis buffer (P0013K, Beyotime, China) on ice for 30 min. The proteins were then scraped off and transferred to centrifuge tubes for centrifugation. The protein concentration was measured by colorimetry (Thermo Scientific, MA) after centrifugation. Then, the loading buffer (P0015, Beyotime, China) was added to the centrifuge tube, which was boiled for 10 min at 100 °C. Twenty micrograms of protein was added to the lane for electrophoresis and then transferred to a polyvinylidene fluoride membrane (IPVH00010, Millipore, USA). The membranes were then incubated with 5% skim milk. Primary antibodies against Akt (4691L, Cell Signaling, China), PI3K (ab227204, Abcam, U.K.), Col-1 (14695-1-AP, Proteintech, China), OCN (ab93876, Abcam, U.K.), ALP (ab108337, Abcam, U.K.) and OPN (22952-1-AP, Proteintech, China) were incubated with the blots overnight at 4 °C. The membranes were washed three times with PBS and then incubated with secondary antibodies at room temperature for 2 h. To visualise the bands, a chemiluminescence kit (Thermo Scientific, MA) was used.
8
QKF Extract Inhibits Inflammation
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Herbal compounds of QKF were provided by a pharmacy of Jilin Provincial Hospital of Traditional Chinese Medicine (Changchun, China). All of the crude drugs (98 g, two-thirds are used clinically) were extracted in 1000 ml of distilled water three times (100°C, 1 h each time) to obtain the aqueous extract. The extracts were centrifuged at 3,500 rpm for 15 min, and the supernatant was freeze-dried to obtain the powdery extract of QKF, with a yield of 20% (13 g) for further experiments. According to dose translation of animal studies, the medium treatment concentration of a mouse is approximately equal to 3 g/kg/day; the low and high treatment concentrations are approximately equal to 1.5 g/kg/day and 6 g/kg/day, respectively. Streptozotocin (STZ) was purchased from Sigma-Aldrich (Shanghai, China). Antibodies against p-PI3K (AF3241, Affinity Biosciences, China), PI3K (ab227204, Abcam, USA), p-Akt (4058, Cell Signaling Technology, USA), Akt (ab179463, Abcam, USA), p-NF-κB (3033, Cell Signaling Technology, USA), NF-κB (ab16502, Abcam, USA), IL-6 (ab208113, Abcam, USA), IL-1β (ab254360, Abcam, USA), TNF-α (8184, Cell Signaling Technology, USA), IKBKB (15649-1AP, Proteintech, China), and GADPH (60004-1-1g, Proteintech, China) were used in this study.
9
PD-1 Signaling Pathways in Murine PBLs
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PD-1 downstream signaling molecules in PBL from mice were measured by Western blot analysis at day 7 of post-infection. The primary antibodies were PI3K (ab227204, 1:1000, Abcam), p-PI3K (ab182651, 1:1000, Abcam), AKT (#9272, 1:1000, Cell Signaling Technology, USA), p-AKT (Ser473) (#9271, 1:1000, Cell Signaling Technology), ERK (#4695S, 1:1000, Cell Signaling Technology), p-ERK (Thr202/Tyr204) (#9101, 1:1000, Cell Signaling Technology), mTOR (ab2732, 1:1000, Abcam), p-mTOR (ab84400, 1:1000, Abcam), and β-actin (60008-1-lg, 1:15000, Proteintech), which was used as an internal control. The secondary antibodies were HRP-conjugated affinipure goat anti-mouse IgG (H+L) (SA00001-1, 1:8000, Proteintech) and HRP-conjugated affinipure goat anti-rabbit IgG (H+L) (SA00001-2, 1:8000, Proteintech).
10
Analysis of Protein Expression in OM-MSCs and Brain Tissue
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Proteins were extracted from OM-MSCs or brain tissue using a total protein extraction kit (P0033; Beyotime Biotechnology). Protein concentration was determined with a BCA Protein Assay Kit (P0010; Beyotime Biotechnology). Protein extracts were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked and incubated with the indicated primary antibodies against P21(1: 1000; 10355-1-AP; Proteintech), P53(1: 2000; 10442-1-AP; Proteintech), P16INK4A (1: 1000; 10883-1-AP; Proteintech), LC3 (1: 1000; 18725-1-AP; Proteintech), P62(1: 2000; 18420-1-AP; Proteintech), Beclin-1(1: 3000; 11306-1-AP; Proteintech), PTBP1 (1:3000; 12582-1-AP; Proteintech), PI3K (1: 1500; ab227204; Abcam), p-PI3K (1: 750; ab182651; Abcam), and actin (mouse, 1:5000; 66009-1-Ig; Proteintech) at 4°C overnight. After washing, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1: 5000; anti-mouse or anti-rabbit IgG; SA00001-1; SA00001-2; Proteintech). The membranes were visualized using an ECL detection kit (Bio-Rad, Munich, Germany).
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