MDA-MB-231 cells were grown to confluence in a T-75 cell culture flask. Cells were treated with 10 mL of fresh media containing 10% FBS, with or without an additional 100 ng/mL EGF for 10 minutes. Flasks were lysed in 4.5 mL of lysis buffer, comprised of 50 mM Tris (pH 7.4), 0.15 M NaCl, 0.1% Triton-X, with protease and phosphatase inhibitors as described above. 1 mL of lysate was immunoprecipitated with 10 μL agarose beads (Thermo Fisher Scientific) with either 5 μL rabbit IgG (ab6718, Abcam), 10 μL anti-EGFR (D38B1, Cell Signaling), or with 10 μL anti-EGFR pre-conjugated to sepharose beads (5735S, Cell Signaling). Lysates were precipitated on beads overnight at 4°C on a rotating platform and spun down. Beads (with protein) were washed 3× with TBS, and boiled in 70 μL of DiH2O and sample buffer. Blotting was done as described above for pEGFR (ab32430, Abcam) and integrin α6 (ab134565, Abcam).
Ab32430
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab32430 is a laboratory equipment product offered by Abcam. It is a device used for scientific research and analysis purposes. The core function of this product is to provide a specific capability required in a laboratory setting. Further details on the intended use or features of this product are not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ab52894, by Abcam (4 mentions)
Ab197772, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Agarose beads, by Thermo Fisher Scientific (1 mentions)
Ab6718, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Phospho p44 42 mapk erk 1 2 t202 y204, by Cell Signaling Technology (1 mentions)
Go6983 hy 13689, by MedChemExpress (1 mentions)
Pan akt, by Cell Signaling Technology (1 mentions)
Ab32199, by Abcam (1 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
Phospho akt thr308, by Cell Signaling Technology (1 mentions)
Ab2893, by Abcam (1 mentions)
P pdk1, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Df3645, by Abcam (1 mentions)
Ab243895, by Abcam (1 mentions)
Sc 74543, by Santa Cruz Biotechnology (1 mentions)
Ab223500, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
7 protocols using ab32430
1
Immunoprecipitation of EGFR Signaling
2
EGFR and Phospho-EGFR Signaling Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies that recognize TSPAN8 (ab70007, 1:1000), HA (#3724, 1:5000), EGFR (ab52894, 1:8000) and phospho-EFGR Y1068 (ab32430, 1:8000) were purchased from Abcam (Cambridge, UK). Anti-SOX9 (#82630, 1:1000), β-actin (#4970, 1:5000), phospho-p44/42 MAPK (Erk 1/2) T202/Y204, rabbit horseradish peroxidase (HRP)-linked (#7074, 1:5000) and mouse HRP-linked (#7076, 1:5000) antibodies were purchased from Cell Signaling Technology (Massachusetts, USA). Anti-ERK1/2 (16443–1-AP, 1:1000) and phospho-AKT S473 (66444–1-lg, 1:1000) were purchased from Proteintech Group, Inc (Chicago, USA). Anti-phospho-PKCα S657 (sc-377565, 1:100) was purchased from Santa Cruz Biotechnology (Texas, USA).
Recombinant human EGF (rhEGF, PHG0311L) was purchased from Gibco (New York, USA). D-luciferin sodium salt (P1043) was purchased from Promega (Wisconsin, USA).
Gefitinib (HY-50895), Erlotinib (HY-50896), AG1478 (HY-13524), Compound 26 (HY-18296), GDC-0994 (HY-15947) and GO6983 (HY-13689) were purchased from MedChemExpress (MCE) Company (Shanghai, China).
Recombinant human EGF (rhEGF, PHG0311L) was purchased from Gibco (New York, USA). D-luciferin sodium salt (P1043) was purchased from Promega (Wisconsin, USA).
Gefitinib (HY-50895), Erlotinib (HY-50896), AG1478 (HY-13524), Compound 26 (HY-18296), GDC-0994 (HY-15947) and GO6983 (HY-13689) were purchased from MedChemExpress (MCE) Company (Shanghai, China).
3
Antibody Profiling of AKT Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies against pan-AKT (#4691, 1:1000), phospho-AKT Thr308 (#13038, 1:1000), phospho-AKT Ser473 (#4060, 1:1000), AKT1 (#2938, 1:1000), AKT2 (#3063, 1:1000), AKT3 (#14982, 1:1000), PDK1 (#13037, 1:1000) and p-PDK1 (#3438, 1:1000) were from Cell Signaling Technology (Danvers, MA, USA). Antibodies against γ- H2AX (ab2893; 1:1000), PTEN (ab32199; 1:10000), EGFR (ab32430; 1:5000) were from Abcam (Cambridge, MA, USA). Antibodies against flag (F1804, 1 mg/mL; 1:1000) and beta-tubulin (T5201; 1:5000) were from Sigma-Aldrich (St. Louis, MO, USA).
4
Quantitative Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The extraction of overall proteins of tissues and cells was made with FLAG lysis buffer with a protease inhibitor cocktail. A BCA kit was used to measure protein concentrations. Proteins were electrophoresis, transferred, and blocked, then incubated with the diluted primary antibodies at 4 °C overnight. PSMD3 (Affinity biosciences, DF3645, 1:1000), ILF3 (Abcam, ab92355, 1:1000), ERBB3 (Affinity biosciences, DF6144, 1:1000), p-EGFR (Abcam, ab32430, 1:1000), EGFR (Abcam, ab32077, 1:1000), and GAPDH (Cell signaling, 21185, 1:1000) were all used in this study. Horseradish peroxidase-labeled secondary antibodies was adopted to incubate the membranes after extensive washes, and a strengthened chemiluminescent kit with a ChemiDoc XRS testing system was adopted to test signals. ImageJ software was adopted to analyze the signals.
5
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fifty micrograms (50ug) of cell protein were extracted from HBE cells according to previous procedures (23 (link)). In brief, isolated protein from cells or lung tissues were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. Then, the PVDF membrane was incubated bated with primary antibody for 12 hours and next incubated with Horseradish Peroxidase (HRP) conjugated secondary antibody. The following antibodies were used to determine the levels of ITGB4 (ab197772, Abcam, USA), EGFR (ab52894, Abcam, USA), p-EGFR (ab32430, Abcam, USA) and IRF-1 (ab243895, Abcam, USA). GAPDH (ab8245, Abcam, USA) was used as corresponding controls as indicated.
6
Protein Expression Analysis in HBE Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted from HBE cells according to previous procedures 18 . The following antibodies were used to determine the expression of corresponding protein: ITGB4 (ab197772, Abcam, USA), EGFR (ab52894, Abcam, USA), p-EGFR (ab32430, Abcam, USA), ERK1/2(ab184699, Abcam, USA), p-ERK1/2(ab223500, Abcam, USA), c-Jun (sc-74543, Santa Cruz, USA), p-c-Jun (sc-822, Santa Cruz, USA). Lamin-β1(ab133741, Abcam, USA) and β-actin (ab8226, Abcam, USA) were used as corresponding controls, respectively.
7
BEAS-2B Cell Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BEAS-2B cells were lysed with RIPA buffer, with the total protein extracted. The protein per lane was loaded onto a 10% SDS-PAGE and transferred to the PVDF membranes (GE Healthcare, USA). The membranes were probed with primary antibodies against EGFR (ab52894, Abcam, Cambridge, UK), p-EGFR (ab32430, Abcam), HSPA6 (ab212044, Abcam), and GAPDH (ab8245, Abcam) overnight at 4°C. Then, the membranes were rinsed three times and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature, and HRP substrates were added to visualize the protein bands. The housekeeping gene GAPDH was used as a control for normalization. Densitometric analysis of immunoblots was performed using ImageJ software (USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!