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NOTE: This cell line is 3xFLAG tagged at the C-term of the p105 NFKB1 precursor protein - the p50 C-terminally cleaved mature form does not contain the 3xFLAG peptide.

NOTE: This construct has two silent mutations.

Doxycycline Induction of NFKB1 Cell line

Date: 09/13/17

Cell lines: HEK293 NFKB1_3xFLAG (treated with Zeocin)

see TF 3xFLAG Flp-In constructs

Induction Grow cells in four 75cm² Cell Culture Flasks

When culture reaches >70% confluence, change to induction medium containing Doxycycline (Dox) in concentrations of 0, 1, 5 or 10ng/ml

Make fresh Dox stock (SIGMA D9891)

Dox stock: 5mg/ml = 5000ug/ml

Make 1:500 dilution of the stock (10ul of 5mg/ml Dox + 4990ul of DMEM): 10ug/ml (working stock)

Final concentration of the induction medium: 0, 1ng/ml, 5ng/ml or 10ng/ml

Will need 10ml of induction medium for each cell line (40ml total @ each concentration for all cell lines; make 50ml should be enough)

Make media using working stock (10ug/ml)

0 (no Dox)

1ng/ml = 1:10,000 (10ul of 10ug/ml Dox in 50ml of medium)

5ng/ml = 1:2000 (25ul of 10ug/ml Dox in 50ml of medium)

10ng/ml = 1:1000 (50ul of 10ug/ml Dox in 50ml of medium)

Incubate O.N. before harvesting (20hr induction this time)

Harvesting Remove media and wash the flask with 10ml of DPBS

Add 1.8ml of DPBS; Release the cells using cell scrapers and transfer to a 2ml eppendorf tube with a 2ml serological pipet; Keep on ice

After collecting all samples, spin @ 1k rcf, 4°C for 5’ (Eppendorf Centrifuge 5417R)

Resuspend in 0.5ml extraction buffer with protease inhibitors

(Extraction buffer:  300mM NaCl 20mM HEPES, pH7.4, 0.5% Tritonx-100)

Sonicate 5x 2 sec @ 2 Amp; Record energy output:

{NFKB1 0 =100J, NFKB1 1 = 107J, NFKB1 5 =104J, NFKB1 10 = 106J}

Spin @ 14k rpm, 4°C for 10’ (Eppendorf Centrifuge 5417R)

Transfer the supernatant to fresh tubes (“Sup”)

Resuspend each pellet in 0.5ml of 1.1x LDS by pipetting and vortex; Incubate @70°C for 5’

Spin @ 20k rcf, 25°C for 5’

Keep the supernatant for Western analysis (“Pellet”)

Western Do Bradford Assay of total protein concentration on each sup fraction:

Load 25ug of total protein of each sup fraction and load same vol. of corresponding pellet fraction

Load 25ug of total protein from the supernatant fraction of HEK293 cells (parental cell line); use it as a negative control

Add 50mM DTT (final concentration) to each sample

Heat samples @ 75°C for 10’

Load 25ug of each sample on 15-well 4-12% Bis-Tris gel

Gel:

1. Marker_5ul
2. HeLa cell lysate (NFkB control)
3. NFKB1+Zeocin_0_Sup
4. NFKB1+Zeocin_Dox1_Sup
5. NFKB1+Zeocin_Dox5_Sup
6. NFKB1+Zeocin_Dox10_Sup
7. NFKB1+Zeocin_Dox10_Sup
8. space
9. NFKB1+Zeocin_0_Pellet
10. NFKB1+Zeocin_Dox1_Pellet
11. NFKB1+Zeocin_Dox5_Pellet
12. NFKB1+Zeocin_Dox10_Pellet
13. NFKB1+Zeocin_Dox10_Pellet
14. LD401_3xFLAG (FLAG control)
15. Marker_2ul

Wet transfer 70V for 1.5h

Block in TBST/5% milk @ RT for 2hr

Primary Ab: mouse anti-NFKB1 antibody (ThermoFisher MA515870), 1:1000 (4°C overnight)

Secondary Ab: HRP linked anti-mouse IgG, 1:10,000 (RT for 1hr)

Strip membrane and probe with anti-FLAG antibody 

Anti-FLAG (Sigma F3165) 

Primary Ab: Mouse anti-FLAG antibody, 1:2,000 (4°C overnight)

Secondary Ab: ECL anti-mouse HRP 1:10,000 (RT for 1hr)

NOTE: These two blot may not correlate The band at ~100kDa that appears to be Dox-dependant may be background. Aligning the images does not show perfect agreement (although nearly). Many of the same bands are in the LD401 control, and Hua's recent experiment failed to reproduce the result. This will be followed up. Present interpretation is that the 3xFLAG tag may be cleaved from the protein, though it is indeed Dox-induced.